Metabolomics: The Basics

David Wishart
Depts. Comp. Sci and Bio. Sci.
University of Alberta
david.wishart@ualberta.ca
July 16, 2005, 8th Banff Symposium
The Pyramid of Life

Metabolomics
1400
Chemicals

Proteomics
2500 Enzymes

Genomics
25,000 Genes
 Metabolomics

Perturbation Primary Molecules

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Secondary Molecules

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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
GrowthinMetabolomics

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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 > 1µ M
 Why 1 µ 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 µ 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 • Very fast
• Requires no work up • Very sensitive
or separation • Allows analysis or
• Allows ID of 300+ ID of 3000+ cmpds
cmpds at once at once
• Good for CHO’s • Not quantitative
• Not sensitive • Not good for CHOs
• Needs MS or 2D • Requires work-up
NMR for positive ID • Needs NMR for ID
 2 Routes to Metabolomics

ppm 7 6 5 4 3 2 1

Quantitative Chemometric (Pattern)

Methods Methods
25
PC2
20
TMAO
creatinine 15
hippurate
allantoin creatinine taurine citrate 10 ANIT
5
hippurate urea 2-oxoglutarate 0
water
succinate -5
fumarate
-10
Control
-15
ppm 7 6 5 4 3 2 1
-20 PAP
PC1
-25
-30 -20 -10 0 10
 Quantitative vs. Chemometric
• Identifies compounds • No compound ID
• Quantifies compds • No compound conc.
• Concentration range of 1 • No compound
µ M to 1 M concentration range
• Handles wide range of • Requires strict sample
samples/conditions uniformity
• Allows identification of • Allows identification of
diagnostic patterns diagnostic patterns
• Limited by DB size • Limited by training set
 Principles of Quantitative
Metabolomics

Mixture

Compound A

Compound B

Compound C
Quantitative Metabolomics with
Eclipse
 Sample Compound List
• (+)-(-)-Methylsuccinic Acid • DL-Carnitine • L-Isoleucine
• 2,5-Dihydroxyphenylacetic Acid • DL-Citrulline • L-Lactic Acid
• 2-hydroxy-3-methylbutyric acid • DL-Malic Acid • L-Lysine
• 2-Oxoglutaric acid • L-Methionine
• Ethanol
• 3-Hydroxy-3-methylglutaric acid • L-phenylalanine
• Formic Acid
• 3-Indoxyl Sulfate • L-Serine
• Fumaric Acid
5-Hydroxyindole-3-acetic Acid • L-Threonine
• • Gamma-Amino-N-Butyric Acid
• L-Valine
• Acetamide
• Gamma-Hydroxybutyric Acid • Malonic Acid
• Acetic Acid
• Gentisic Acid • Methylamine
• Acetoacetic Acid
• Glutaric acid • Mono-methylmalonate
• Acetone
• Glycerol • N,N-dimethylglycine
• Acetyl-L-carnitine
• Glycine • N-Butyric Acid
• Alpha-Glucose
• Glycolic Acid • Pimelic Acid
• Alpha-ketoisocaproic acid
• Hippuric acid • Propionic Acid
• Benzoic Acid
• Pyruvic Acid
• Betaine • Homovanillic acid
• Salicylic acid
• Beta-Lactose • Hypoxanthine
• Sarcosine
• Citric Acid • Imidazole
• Succinic Acid
• Creatine • Inositol Sucrose
•
• Creatinine • isovaleric acid • Taurine
• D(-)Fructose • L(-) Fucose • trans-4-hydroxy-L-Proline
• D-(+)-Glyceric Acid • L-alanine • Trimethylamine
• D(+)-Xylose • L-asparagine • Trimethylamine-N-Oxide
• Dimethylamine • L-aspartic acid • Urea
• DL-B-Aminoisobutyric Acid • L-Histidine
• L-homocitrulline
 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
PC2
PAP 20

15

10 ANIT
5
ANIT 0

-5

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

Principal Component Analysis

 Disease Diagnosis via NMR
(140+ Detectable Conditions)
• Adenine
Phosphoribosyltransferase
Deficency
• Adenylosuccinase Deficiency
• Alcaptonuria
• α -Aminoadipic Aciduria
• β -Aminoisobutyric Aciduria
• α -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 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 • 96% sensitivity and 100%
• 11 methylmalonic aciduria
• 11 cystinuria specificity in ID of
• 6 alkaptonuria abnormal from normal by
• 4 glutaric aciduria I
• 3 pyruvate decarboxylase deficiency metabolite concentrations
• 3 ketosis
• 3 Hartnup disorder
• 95.5% sensitivity and 92.4%
• 3 cystinosis specificity in ID of disease
• 3 neuroblastoma
• 3 phenylketonuria or condition by
• 3 ethanol toxicity
characteristic metabolite
• 3 glycerol kinase deficiency
• 3 HMG CoA lyase deficiency concentrations
• 2 carbamoyl PO4 synthetase deficiency
• 120 sec per sample

Clinical Chemistry 47, 1918-1921 (2001).

 Applications in
Metabolite Imaging
Lactate N-acetyl-aspartate

Glutamate

Citrate
Alanine
 Absent
Normal
Below Normal
Above Norrmal

Patient 9
Patient 8
Patient 7
Patient 6
Patient 5
Patient 4
Patient 3
Patient 2
Patient 1

Patient 15
Patient 14
Patient 13
Patient 12
Patient 11
Patient 10
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 Acetone, Lactate, Ethanol, Succinate, Citrate, Glucose, Urea
(chemical) TMAO, Dimethylamine, Taurine Allantoin (urine & serum)
(urine & serum)

Kidney (Human) Graft Dysfunction TMAO, Dimetheylamine

Lactate, Acetate, Succinate, Glycine,
Alanine, (urine)

Kidney (Rat) Graft Dysfunction TMAO, Citrate, Lactate,

Reperfusion Injury Dimetheylamine, Acetate (urine)

Kidney (Rat) Reperfusion Injury TMAO, Allantoin (serum)

(ischemia)

Kidney (Human) Graft Dysfunction TMAO, Alanine, Lactate,

CsA toxicity Citrate (urine & serum)

Kidney (Mouse) Nephrectomy Methionine, Citrulline, Arginine, AlanineSerine

(urine & serum) (serum)

Kidney (Mouse) Nephrectomy Guanidinosuccinate, Guanidinoacetate (urine)

Guanidine, Creatinine,
Guanidinovalearate,
(urine & serum)

Kidney (Human) Acute Rejection Nitrates, Nitrites, Nitric oxide

metabolites (urine)
Applications In Transplantation
Organ Condition Metabolite(s) Increased Metabolite(s) Decreased 22

Liver (Rat) Reperfusion Injury Citrate, Succinate, Ketone bodies (good Citrate, Succinate, Ketone bodies
function) (poor function)

Liver (Human) Ischemia Methylarginine

Dimethylarginine
(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 N-acetylaspartate,

Failure 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 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.
 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...