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Untargeted Metabolomic
Profiling in Inborn Errors of
Metabolism
V. Reid Sutton, MD
Professor, Molecular & Human Genetics
Baylor College of Medicine
Medical Director, Biochemical Genetics
Laboratory
testing
prevention
research
education
solutions
Disclosure
A fixed portion of my salary is paid by Baylor Genetics Laboratories but compensation is not tied to laboratory revenue
Outline
• Common practice and limitations of current routine testing for IEMs
• Global Metabolomic Assisted Pathway Screen (Global MAPS®) - Methods
• Validation for common IEMs
• Confirmation of DNA variant pathogenecity
• Discovery of Novel Biomarkers
CURRENT RECOMMENDATIONS FOR INTELLECTUAL DISABILITY EVALUATION
AAN Recommendations for Intellectual Disability (2011)
• Screening for inborn errors of metabolism (IEMs) in children with GDD/ID has a yield of between 0.2% and 4.6%, depending on the presence of clinical indicators and the range of testing performed (Class III).
• Testing for congenital disorders of glycosylation has a yield of up to 1.4%, and testing for creatine disorders has a yield of up to 2.8% (Class III).
van Karnebeek CDM et al., Mol Genet & Metab 111:428-38, 2014
Current Challenges
• For undifferentiated phenotypes, such as intellectual disability, seizures, recurrent vomiting, failure to thrive etc. many different tests are needed
• Various fluid types (blood, urine, cerebrospinal fluid) may be needed for diagnosis
• Cost for multiple tests may be prohibitive and many are rare, so no good way to tier testing
Methods/Tests
• HPLC – amino acids • GC/MS – organic acids • MS/MS
– Acylcarnitines – Newborn screening – Individual specialized tests
• Purines & Pyrimidines • Creatine & guanidinoacetate • Pyridoxine responsive seizure panels • Bile acids • CSF Neurotransmitters • Etc!
Rationale for Metabolomic Approach
9
DNA
RNA
Proteins
threonine
N H 2
O H O
O H
glucose
O H
O H
O H
O H
O
O H
cholesterol
Mechanistic Insight into Phenotype
Biochemicals
Biochemistry Advantages
Any sample type
Actionable
Condensed & information rich
Bridge between genome & phenotype
METHODS
Pioneering the emerging field of global biochemical pathway analysis for biomarker discovery and the development of innovative diagnostic tests • Founded in 2000
• Over 150 employees worldwide with expertise in biochemistry, mass spectrometry and software development
• 54,000 sq. ft. facility in Research Triangle Park, NC and Sacramento
• CLIA-certified lab onsite
Metabolon, a global leader in metabolomics
Biomarkers Mechanistic Understanding Drug MoA Cellular Characteristics
Interpretation
Biochemical Extraction
UHPLC (HILIC)-MS/MS
UHPLC-MS/MS (+ESI) Late/Lipid
UHPLC-MS/MS (+ESI) Early/Polar
UHPLC-MS/MS (-ESI) Compound ID
Library Search RT, Mass, MS/MS
QA/QC
Data Reduction
Statistical Analysis
Pathway Visualization: Metabolync™ plugin to Cytoscape developed to overlay analyte findings onto metabolic pathways
Biomarkers Mechanistic Understanding Drug MoA Cellular Characteristics
Interpretation
Biochemical Extraction
Compound ID
Library Search RT, Mass, MS/MS
QA/QC
Data Reduction
Statistical Analysis
UHPLC (HILIC)-MS/MS
UHPLC-MS/MS (+ESI) Late/Lipid
UHPLC-MS/MS (+ESI) Early/Polar
UHPLC-MS/MS (-ESI)
Enrich 50-1500 Da small molecule analytes
Biomarkers Mechanistic Understanding Drug MoA Cellular Characteristics
Interpretation
Biochemical Extraction
Compound ID
Library Search RT, Mass, MS/MS
QA/QC
Data Reduction
Statistical Analysis
•Q exactive MS/MS - Orbitrap based MS/MS (Thermo) •Accurate to 1 ppm vs 100 ppm for quadrupole analyzers •Cost ~2X quadrupole
UHPLC (HILIC)-MS/MS
UHPLC-MS/MS (+ESI) Late/Lipid
UHPLC-MS/MS (+ESI) Early/Polar
UHPLC-MS/MS (-ESI)
Biomarkers Mechanistic Understanding Drug MoA Cellular Characteristics
Interpretation
Biochemical Extraction
Compound ID
Library Search RT, Mass, MS/MS
QA/QC
Data Reduction
Statistical Analysis
•Library of ~2500 human metabolites
UHPLC (HILIC)-MS/MS
UHPLC-MS/MS (+ESI) Late/Lipid
UHPLC-MS/MS (+ESI) Early/Polar
UHPLC-MS/MS (-ESI)
Biomarkers Mechanistic Understanding Drug MoA Cellular Characteristics
Interpretation
Biochemical Extraction
Compound ID
Library Search RT, Mass, MS/MS
QA/QC
Data Reduction
Statistical Analysis
Pathway Visualization: Metabolync™ plugin to Cytoscape developed to overlay analyte findings onto metabolic pathways
UHPLC (HILIC)-MS/MS
UHPLC-MS/MS (+ESI) Late/Lipid
UHPLC-MS/MS (+ESI) Early/Polar
UHPLC-MS/MS (-ESI)
17
Metabolon’s Approach to Metabolomics - a Comprehensive Survey of the Metabolome
18 Baylor Genetics
Extensive Metabolite
Library
High resolution biochemistry surveys central metabolism &
peripheral pathways that drive
attributes of phenotype.
1.14.15.4
1.3.1.30
progesterone
11b-hydroxy
progesterone
PHENYLALANINE
TYROSINE
METABOLISM
Microbiome
Microbiome
phenylacetyl-CoA
phenylacetate
glutamine
3-iodotyrosine
3-iodotyrosine3,5-diiodo-L-tyrosine 1.11.1.8
pregnanolone1.1.1.50
3,5-diiodo-L-tyrosine THYROID
HORMONE
SYNTHESIS
fumarate
4.2.1.2
2.3.1.9
1.3.5.1
1.1.1.37
malate
4.1.1.53
2-phenylethylamine
1.4.3.4
2-phenylacetaldehyde
1.2.1.5
(4-hydroxyphenyl-)
acetaldehyde
1.2.1.5
Microbiome
4-hydroxyphenylacetate
1.14.16.2
Microbiome
4.1.1.28
2.6.1.51.4.3.2
tyrosine
4-hydroxyphenylpyruvate
1.13.11.27
homogentisate
1.13.11.5
1.4.3.4
Microbiome
phenol sulfate
1.14.16.1
1.4.3.4
tyramine
1.11.1.8
STEROID
HORMONE
BIOSYNTHESIS
dihydoxyphenylalanine
(L-DOPA)
Microbiome
SULF
phenol
1.1.1.213
tetrahydrodeoxy
corticosterone
5beta-pregnane-3,20-dione
allopregnane-
21-ol-3,20-dione
1.3.1.30
20a,22b-dihydroxy
cholesterol
2.6.1.1
phenyllactate
(PLA)
phenylpyruvate
3-hydroxy-
2-phenylpropanoate
Microbiome
phenylalanine
1.13.11.27
1.4.3.2
3-hydroxyphenylacetate
1.11.1.8
1.11.1.8
5a-pregnane-3,20-dione
2-hydroxyphenylacetate
2,2-dimethylsuccinate
(Food
Additive)
6.2.1.5
5.3.3.-
TCA
CYCLE
6.2.1.4
succinate
3-b-HSD / SDI
1.14.15.4
1.14.15.4
1.14.99.9
1.14.99.10
1.14.15.4
1.1.1.149
thyroxine (T4)
3,5,3'-triiodothyronine
(T3)
1.1.1.213
1.1.1.53pregnanediol
1.14.99.10
3-b-HSD / SDI
7alpha-hydroxy
pregnenolone 1.14.13.100
3-b-HSD / SDI
17a,21-dihydroxypreg-
nenolone
1.14.99.10
3-b-HSD / SDI
1.14.15.4
11b,17a,21-trihydroxy
pregnenolone
cortexolone
20a-dihydro
progesterone
17-hydroxy
progesterone
1.1.1.149
1.14.99.9
3-b-HSD / SDIdeoxycorticosterone
21-hydroxy
pregnenolone
1.3.1.3
1.14.99.10
1.14.99.10
pregnenolone
2.8.2.23.1.6.2
1.14.99.9
17a-hydroxypregnenolone
1.14.15.6
4-methylpentanal
20alpha-hydroxy
cholesterol
1.14.15.6
22b-hydroxy
cholesterol
1.14.15.6
pregn steroid
monosulfate
3,4-dihydroxyphenylacetate
1.2.1.5
2.1.1.28
2.1.1.6
3-methoxy-
4-hydroxyphenyl
acetaldehyde
noradrenaline
2.1.1.6
dopamine
1.14.17.1
1.4.3.4
2.1.1.6
3,4-dihydroxy
phenylacetaldehyde
1.2.1.3
3-methoxytyramine
1.4.3.4
homovanillate
(HVA)
1.14.11.8
3-hydroxy-
6-N-trimethyllysine
4-N-trimethylamino-
butyraldehyde 1.2.1.474.1.2.-
Microbiome
carnitine
3-dehydrocarnitine
1.14.11.1
deoxycarnitine
CARNITINE
BIOSYNTHESIS
2.1.1.43
Protein-Lysine
3.4.-.-
1-methyl-
histidine
phospho-
threonineProteinogeneic
Amino Acid
Protein
Degradation
5-hydroxylysine
3-hydroxyproline
3-methyl-
histidine
N6-acetyllysine
Protein-
6-N-trimethyl-
lysine
Protein-
6-N-dimethyl-
lysine
Protein-
6-N-methyl-lysine2.1.1.432.1.1.43
3-phosphoserine
PROTEIN
DEGRADATION
cis-4-hydroxyproline
acetyl-CoA
trans-4-
hydroxyproline
N6-trimethyl-
lysine
assymetric
dimethylarginine
(ADMA)
dimethylarginine
(SDMA +
ADMA)
5.3.3.- 2.3.1.7
3-hydroxyisovalerate
3-hydroxyisovaleryl-CoA3-methylcrotonylglycine
N-acetyl-amino
acids
Dipeptide
3-methylglutarylcarnitine
(C6)
Polypeptide
3-methylglutarateacetoacetate
3-hydroxy-3-methylglutarate
3-methylglutaconyl-CoA
4.2.1.18
3-hydroxy-
3-methylglutaryl
CoA
4.1.3.4
2-butanone
3.1.2.20
3-methylglutaconate3-methylglutaryl-CoA
succinyl CoA
2.8.3.5 3.1.2.20 5.3.3.-
hydroxyisovaleroyl
carnitine
1.1.1.178
3.1.2.20
2.3.1.16
2-methyl-
3-hydroxybutyryl-CoA
2-methylacetoacetyl-CoA
3.1.2.20
1.1.1.35
5.3.3.-
2-methylacetoacetate
saccharopine
1.5.1.9
glyceraldehyde
4.1.2.13
2.7.1.3
fructose
1-phosphate
1.5.1.8
2.6.1.22
2-keto-6-aminocaproate
SPON
1.4.3.14
1.2.1.27
5.1.99.1
(S)-methylmalonyl-CoA
2.3.1.7
propionyl CoA
6.4.1.3
2.3.1.13
propionylglycine
methylmalonic
acid
semialdehyde
3.1.2.20
1.2.1.31.2.3.1
methylmalonate
SORBITOL
AND
GLYCEROL
METABOLISM
dihydroxyacetone
2.7.1.31
1.1.1.6
5.3.1.1
2.7.1.29
dihydroxyacetone
phosphate
(DHAP)
glycerol1.5.3.7
2,3,4,5-tetrahydropiperidine-
2-carboxylate
delta-piperideine
-2-carboxylate
1.5.1.21
5-aminovalerate
Microbiome
(R)-methylmalonyl-CoA
glutarate
semialdehyde
pipecolate
3-hydroxyisovalerate
isovalerylsarcosine
2.3.1.7
methylsuccinate
mesaconate
(methylfumarate)
4.2.1.17
isovalerylcarnitine
BCKD
6.4.1.4 2.3.1.13
3-methylcrotonyl-CoA
isovaleryl-CoA
2.6.1.42
4-methyl-
2-oxovalerate
1.3.8.4
leucine
isovalerylglutamate
3-hydroxyisoheptanoate
5.3.3.-
4-hydroxyisovalerate
isovalerylalanine
isovaleryglucuronide
2.3.1.13
5.3.3.-
isovalerylglycine3.1.2.20
3-methyl-1-
hydroxybutyl-ThPP
1.3.8.7
S-(3-methylbutanoyl)-
dihydrolipoamide-E
1.3.8.64.2.1.17crotonoyl CoA
1.3.99.7glutaconyl-CoA
GLYCOLYSIS
1.2.1.3
2.6.1.2
2.6.1.44
2.6.1.39
SPON
2-aminoadipate
semialdehyde
5.3.3.-
1.2.1.31
LYSINE
CATABOLISM
ADR/ ALDR
2-aminoadipate
2.3.1.9
1.1.1.35
3-hydroxybutyryl
CoA
coenzyme A
acetylcarnitine
acetoacetyl-CoA
glutaconate
2.3.1.7
alanine
Microbiome
KGD2-oxoadipate
glutarylcarnitine
(C5)glutaryl CoA
5.3.3.-
Microbiome
glutarate
(pentanedioate)
ethylmalonyl
CoA
3.1.2.20
3-hydroxyisobutyrate
3-hydroxyisobutyryl-CoA
3.1.2.4
3-hydroxy-
2-methylbutyrate
BCAA
METABOLISMethylmalonate
cadaverine
2.3.1.7 2.3.1.13
butyrylglycinebutyrylcarnitine
3.1.3.11
Microbiome
2.7.1.11
fructose-6-phosphate
fructose-1,6-bisphosphate
4.1.2.13
5.3.1.9
5.4.2.5
1.1.1.14
lysine
2.7.1.24
4.1.1.18
sorbitol
dephospho-
coenzyme A
fructose
2.7.1.1
sorbitol
6-phosphate
2.7.1.69
1.1.1.21
propionylcarnitine1.1.1.313-aminoisobutyrate
1.1.1.35
1.3.8.1
2-methylbutyrylcarnitine
(C5)
isovalerate3.1.2.20
?
1.4.3.2
2.3.1.7
alpha-hydroxyisocaproate
2-methylbutyrate
2.3.1.13
tiglate3.1.2.20
2-methylbutyrylglycine
2-hydroxy-3-methylvalerate ?
1.3.8.7
2-methyl-1-
hydroxybutyl-ThPP
3.1.2.20
S-(2-methylbutanoyl)-
dihydrolipoamide
2.6.1.42
1.3.99.12
BCKD
2-methylbutyryl-CoA
tiglyl-CoA
3-methyl-2-oxovalerate
isoleucine
tigloylglycine
4.2.1.17
2.3.1.13
3.1.2.20
3-methylcrotonate
2.3.1.7
tiglyl carnitine
2-ethylacrylyl-CoA
1.3.99.-
2.6.1.42
1.2.1.-
3.1.2.4
(R)-2-methylbutyryl-CoA
2-ethylhydracryyl-CoA
4.2.1.17
3-methyl-2-oxovalerate
2.4.1.1
maltopentaose
maltotetraose
2.4.1.1
maltotriose
2.4.1.1
glucose-1-
phosphate
2.4.1.1
3.1.3.9
glucose
maltose
phosphopantetheine
4.1.1.36
2.7.7.3
4'-phosphopantothenoyl-cysteine
6.3.2.5
2.4.1.1
2.4.1.15.4.2.2
glucose
1-phosphate
2.4.1.1
maltohexaose
methacrylyl-CoA
1.3.99.-
4.2.1.17
2.6.1.42
Isobutyryl-CoA
3-methyl-2-oxobutyrate
BCKD
valine
maltodextrin
(n)
GLYCOGEN
METABOLISM
2.7.1.33
PANTOTHENATE
(VIT B5)
and CoA
METABOLISM
4'-phosphopantothenate
pantothenate
2-ethylhydracrylate
1.2.1.3
6.4.1.3
butyryl CoA
2-ethylmalonic
semialdehyde
1.1.1.31
ROS
cysteine
2.7.1.33
pantethine
5.3.3.-
3.5.1.92pantetheine
L-alloisoleucine
2.3.1.7
?
2-methyl-1-
hydroxypropyl-ThPP
S-(2-
ethylpropionyl)
-dihydrolipoamide-E
isobutyrate 3.1.2.20
alpha-hydroxyisovalerate
2.4.1.11
GDE
UDP-glucose
2.4.1.18
glycogen (n-1
residue)Glycogen
2.3.1.131.3.8.71.3.99.121.3.8.1
2-methylglutaconate
isobutyrylcarnitine isobutyrylglycine
2.7.7.9
5.1.3.2
2.7.7.10
UDP-galactose
GALACTOSE
METABOLISM
2.7.1.1
galactitol
(dulcitol)1.1.1.21
1.1.1.48
1.1.1.17
fructose
GUT / DIETtagatose
3.2.1.23
5.3.1.8
galactose
2.7.1.6fructose-6-phosphate
lactose
5.3.3.-
6'-sialyllactose
galactono-
1,4-lactone
3.1.1.25
galactose
1-phosphate
3.5.99.6
2.7.7.12
2.6.1.16
glucosamine-6-phosphate 2.7.1.1 glucosamine
N-acetylglucosamine2.7.1.59
2.3.1.4
N-acetylglucosamine
6-phosphate
4.1.1.34
beta-keto-
galactonate1.1.1.45
5.5.1.4
Microbiomegalactonate
5.4.2.3
N-acetyl-glucosamine
1-phosphate2.7.7.23
UDP-N-acetylglucosamine
GLYCOSYLATION
5.4.2.8
2.7.7.22
GDP-mannose
2.7.7.13
mannose-1-phosphate
mannose-6-phosphate
4.2.1.47
2.7.1.52
fucose-1-phosphate
1.1.1.271
GTP
FPGT
GDP-fucose
GDP-4-dehydro-
6-deoxy-D-mannose
fucose
Microbiome
2.7.1.12.7.1.7
mannitol
inositol-1-P
mannitol-1-phosphate
3.1.3.22
1.1.1.224
mannose
4.1.3.16
ribose
1-phosphate
erythro-
4-hydroxyglutamate
1.1.1.21
D-ribose 2.7.1.15
erythrose
trans-4-
hydroxyproline
1.1.1.21
erythritol
(S)-3-hydroxy-
3-methylglutaryl-CoA
cis-aconitate
4.2.1.3
4.1.1.6
itaconate
(methylenesuccinate)
citrate
3-hydroxybutyrate
(BHBA)
2-methylcitrate
beta-hydroxypyruvate
acetoacetate4.1.3.4
1.1.1.30
1.1.1.79
1.1.3.15
2.6.1.45
glyoxylate
4-hydroxy-
2-oxoglutarate
1.1.1.27
2.3.3.8
2.3.3.1
Microbiome
oxaloacetate
acetyl CoA
malonyl-CoA
2.3.3.10
1.1.1.81
oxalate
(ethanedioate)
pyruvate
PDC
lactate
2.7.1.165
4.3.1.17
1.1.1.27
MDH
6.4.1.1
4.1.1.32
2.2.1.2
3.1.3.23
2.6.1.52
erythrose
4-phosphate
xylulose
6-phosphonoglucono-
1,5-lactone
1.1.1.10
1.1.1.21
3.1.1.316-phosphogluconate
xylitol (D or L)
ribose
5-phosphate2.7.1.17
sedoheptulose-
7-phosphate
2.2.1.1
xylulose
5-phosphate
5.4.2.7
glyceraldehyde
3-phosphate
ribulose
5-phosphate
PENTOSE
PHOSPHATE
PATHWAY
5.3.1.6
1.1.1.44
5.1.3.1
2.7.1.16ribulose
1.1.1.49
3.1.3.46
1.2.1.12
2.7.1.2
glyceraldehyde-3-phosphate
fructose-2,6
-bisphosphate
glucose-6-phosphate
5.4.2.4
3.1.3.13
2,3-diphosphoglycerate
1.1.1.95
2.2.1.1
3-phospho-hydroxypyruvate
fructose-6-phosphate
2.7.9.2
phosphoenolpyruvate
(PEP)
4.2.1.11
2-phosphoglycerate
5.4.2.11
1,3-biphosphoglycerate
2.7.2.3
3-phosphoglycerate
2.7.1.31
3.1.3.3
3-phosphoserine
glycerate
serine
nicotinic acid
mononucleotide
(NaMN)2.4.2.19
2.7.1.173
2.4.2.12
6.3.4.21
nicotinate
ribonucleoside2.4.2.1
nicotinurate
2.3.1.13
Microbiome
quinolinate
5-phospho-alpha-D-ribose
1-diphosphate
isonicotinamide
6-hydroxynicotinate
nicotinate
2.1.1.1
ISO
nicotinamide 2.4.2.1
ADP-ribose
nicotinamide
riboside
NAADP
1-methylnicotinamide
skatol
5.3.3.-
1.13.11.11
5.3.3.-
skatole sulfate
N-formylkynurenine
1.13.11.52
tryptophan
kynurenine
tryptamine
Indole-3-acetaldehyde
1.4.3.24.1.1.28
1.4.3.4
Microbiome
indolepyruvate
2.1.1.41.3.1.72 2.3.1.87 N-acetylserotonin4.1.1.285-hydroxy-L-tryptophan
serotonin
(5HT)
1.2.3.1
SULF
indole
N1-methyl-
4-pyridone-
3-carboxamide
Microbiome
3-indoxyl
sulfate
5-hydroxyindoleacetaldehyde
serotonin
O-methyltransferase
1.4.3.4
1.14.16.4
5-methoxytryptamine
5.4.99.7
lanosterol
1.14.13.70
4,4-dimethylcholesta-
8,14,24-trienol
24,25-dihydrolanosterol
1.3.1.70
4,4-dimethyl-5a-
cholesta-8,24-dien-3-b-ol
1.3.1.72
2-amino-
muconate1.5.1.-
1.2.1.32
NH3
3-hydroxyanthranilate1.13.11.6
aminocarboxymuconate
semialdehyde 4.1.1.45
2-Aminomuconate
semialdehyde
SPON
3.7.1.3
TRYPTOPHAN
METABOLISM
xanthurenate
3-hydroxykynurenine
alanine indoleacetate
2.7.1.36
mevalonate
3-hydroxy-
3-methylglutaryl
CoA
3.1.2.27
1.1.1.34
3-hydroxy-3-
methylglutarate
SPON
4-(2-aminophenyl)-
2,4-dioxobutanoate 2.6.1.7
kynurenate
2.6.1.7
1.14.13.9
indolelactate
1.2.1.3
3.7.1.3
anthranilate
indolepropionate 3.1.3.5
2.4.99.20
3.2.2.6
2.7.1.22
nicotinamide
ribonucleotide
(NMN)
1.6.1.2
NADPH
NADH
6.3.5.1
glutamate
1.14.14.1
1.13.11.526-hydroxymelatonin
melatonin
N-gamma-acetyl-
N-2-formyl-
5-methoxykynurenamine
N1-methyl-
2-pyridone-
5-carboxamide
1.2.3.11.2.3.1
5-hydroxyindoleacetate
nicotinate
adenine
dinucleotide
(NAAD+)
3.1.3.5
2.7.7.18
3.6.1.9
NICOTINATE
AND
NICOTINAMIDE
METABOLISM
glutamine
2.7.1.23
nicotinamide
adenine
dinucleotide
(NAD+)
5-aminolevulinate
NADP+
2.7.7.1
2.3.1.37
3.6.1.22
glycine
acetoacetyl-CoA
2.3.1.192
3-(4-hydroxyphenyl)lactate
2.3.1.14 phenylacetylglutamine
phenylacetylglycine
epinephrine
2.1.1.6
metanephrine
1.4.3.43.1.2.11
5.2.1.2
4.1.1.4
4-fumaryl-acetoacetate
3.7.1.2
acetone
4-maleyl-acetoacetate
acetoacetate
1.4.3.4
3-methoxy-4-
hydroxyphenylglycolaldehyde
normetanephrine
1.2.1.5
3,4-dihydroxy
mandelaldehyde
3,4-dihydroxyphenethyleneglycol
2.1.1.6
3-methoxy-4-
hydroxyphenylglycol
1.1.1.1
1.1.1.1
geranyl
diphosphate
isopentenyl
pyrophosphate
(IPP)
4.1.1.33
2.5.1.1
trans,trans-farnesyl
diphosphate
2.5.1.21
5.3.3.2
dimethylallyl
pyrophosphate
(DMAPP)
6.4.1.2
2.5.1.10
2.3.1.85
acyl-acp
R-mevalonate
5-diphosphate
MEVALONATE
METABOLISM
2.7.4.2
acetoacetyl
CoA
2.3.3.10
mevalonate
5-phosphate
2.3.1.9
5.4.99.2
3.5.1.9
1.14.13.132squalene (S)-2,3-epoxysqualene
vanillylmandelate
(VMA)
MicrobiomeMicrobiome Microbiome ursodeoxycholate
Microbiome
deoxycholate
L-urobilin
taurodeoxycholate
SECONDARY
BILE
ACIDS
L-stercobilinogen
5.3.3.-
Microbiome
cholate
glycodeoxycholate
Microbiome ?
beta-muricholate
Microbiome
ursocholategamma-muricholate
Microbiometaurolithocholate Microbiome
chenodeoxycholate
taurolithocholate
3-sulfate
Detox
Microbiome
lithocholate
glycolithocholate
glycolithocholate
sulfate
Microbiome
Microbiome
mesobilirubinogen
D-urobilin
urobilinogen
5.3.3.-
6.2.1.7
alpha-muricholate
1.14.15.6
?
beta-muricholate
3.1.2.27
chenodeoxycholoyl-CoA
2.3.1.652.3.1.65
MicrobiomeMicrobiome
1.14.13.97
MicrobiomeMicrobiome
hyocholate
3.2.1.31
6.2.1.73.1.2.27
choloyl-CoA
glycocheno
deoxycholate taurocholate
2.3.1.65
Detoxglycocholenate
sulfate
taurocholenate
sulfate
glycocholate
2.3.1.65
Detox
taurocheno
deoxycholate
1.14.13.15 17a,20a-dihydroxy
cholesterol
1.14.13.15 1.14.13.151.1.1.50
1.1.1.181
4.2.1.1071.1.1.35
2.3.1.176
7-alpha-hydroxy-3-
oxo-4-cholestenoate
1.3.1.3
6.2.1.7
1.1.1.501.14.13.15
BILE
ACID
METABOLISM4-cholesten-2alpha,
12alpha-diol-3-one
7-alpha,26-dihydroxy-
4-cholesten-3-one
7
alpha,24-dihydroxy-
4-cholesten-3-one
1.1.1.35 4.2.1.107 1.17.99.3 6.2.1.75.1.99.4
1.14.13.15
1.14.13.15
1.14.13.15
bilirubin
beta-diglucuronide
bilirubin (E,Z
or Z,E)
bilirubin (Z,Z)
SPON
2.4.1.17
SPON
bilirubin (E,E)
2.3.1.176
1.17.99.3 5.1.99.4
7-a,25-dihydroxycholesterol
(24S)-cholest-5-ene-
3beta,7alpha,24-triol
HEMOGLOBIN
AND
PORPHYRIN
METABOLISM
1.14.99.3
1.1.1.181
7alpha,25-dihydroxy-
4-cholesten-3-one
1.14.13.96
1.1.1.181
1.3.1.24 ROS
biliverdin
1.1.1.181
7a-hydroxy-cholestene-3-one7-alpha,27-dihydroxycholesterol
1.3.1.3
3
beta,7-alpha-
dihydroxy-5-cholestenoate
1.14.99.9
3-beta-hydroxy-5-cholestenoate
1.14.13.100
TDTEDKGEFLSEGGGV
Peptidase
vitamin D3
1.14.13.159
TDTEDKGEFLSEGGGVR
Peptidase
calcidiol
Peptidase
1.14.13.13
SPVPDXVPGSFK
DSDKVDXSXAR
Light
Peptidase
TTDSDKVDXSXA
coproporphyrinogen
I
ADpSGEGDFXAEGGGVRPeptidasePeptidase Peptidase
uroporphyrin
III
4a-carboxy-4b-methyl-
5a-cholesta-8,24-dien-3b-ol
1.1.1.170
1.14.13.72
3-keto-4-methylzymosterol
1.1.1.270
4,4-dimethyl-5a-
cholesta-8-en-3b-ol
TTDSDKVDXSXAR
Peptidase
ATTDSDKVDXSXA
Peptidase
4.1.1.37
fibrinogen
peptide B DEG
Peptidase
ATTDSDKVDXSXAR
Peptidase
FIBRINOGEN
CLEAVAGE
PEPTIDES
3.4.21.5
1.14.13.126
1.14.13.126secalciferol
5a-cholest-8-en-3b-ol
calcitriolcalcitetrol
1.3.1.72zymosterol
CHOLESTEROL
AND
VITAMIN-D
SYNTHESIS
DTGTTSEFXEAGGDXR
fibrinogen
peptide A
Peptidase
Peptidase
Peptidase
fibrinogen
ADTGTTSEFXEAGGDXR
Peptidase
ADSGEGDFXAEGGGVR
porphobilinogen
4.2.1.75
4.2.1.24
uroporphyrinogen-III
2.5.1.61
hydroxymethylbilane
ROS
uroporphyrinogen
I
Uroporphyrin I
SPON
protoporphyrinogen
IX
1.3.3.4
coproporphyrinogen
III
4.1.1.37
1.3.3.3
4.99.1.1
protoporphyrin
IX
hemoglobin/heme
proteins
heme
ROS
SDKVDXSXAROS
Peptidase
coproporphyrin
III
Peptidase
coproporphyrin
I3.1.6.2
2.8.2.2
DSGEGDFXAEGGGVR
25-hydroxycholesterol
1.3.1.72
cholesterol
sulfate
1.14.13.100
1.14.99.38 1.14.13.98
24(S)-hydroxycholesterol
desmosterol
1.14.13.99
1.14.15.6
Cholesteryl
Ester
2.3.1.26
1.14.15.6
1.14.13.151.14.13.100 1.14.13.17 1.1.1.181
1.14.13.17
1.14.13.15
7-alpha-hydroxycholesterolcholest-5-ene-3beta,26-diol
cholesterol
7-dehydrocholesterol
5.3.3.-
1.3.1.21
1.3.1.72 7-dehydrodesmosterol
5.3.3.5
1.3.1.72 1.14.21.6lathosterol
5.3.3.5
2.3.1.43
5.3.3.-
5a-cholesta-
7,24-dien-3b-ol
4a-methylzymosterol
5-phosphoribosyl-
5-aminoimidazole-
4-carboxamide
6.3.2.6
5'-phosphoribosyl-
4-(succinocarboxamide)-
5-amin
5'phosphoribosyl-
5-aminoimidazole-4-carboxylate
4.3.2.2
5-aminoimidazole-
4-carboxamide
5,6-dihydrouracil
3.5.2.2
2.4.2.7
ureidopropionate
3.5.1.6
beta-alaninePYRIMIDINE
METABOLISM
2.4.2.4
deoxyuridine
2.4.2.1
deoxycytidine
3.5.4.5
2.4.2.4
thymine
RNA
2.7.7.6GTP2.7.4.6
1.17.4.1
GDP3.6.1.6
cGMP
2.7.4.8
3.1.4.17
3.6.1.19
4.6.1.2
2.1.2.3
1.2.3.1pyridoxate
4-pyridoxolactone1.1.1.107
3.1.1.27
DIETARY:
Plant
compoent
aspartate
pyridoxamine
2.7.1.35
3.1.3.74
XENOBIOTIC:
Xanthine
Metabolism
3.1.3.74
XENOBIOTIC:
Gut bacterial
conversion
2.1.2.3
5-formamido-1-(5-phosphoribosyl)
imidazole-4-carb
pyridoxal
aspartate
PLP
pyridoxamine
phosphate1.4.3.5
XENOBIOTIC:
Tobacco
Metabolite
3.6.3.14
dATP2.7.7.7 2.7.4.6
ATP2.7.7.6
1.4.3.5
pyridoxine
phosphate
2.7.1.35
XENOBIOTIC:
Drug
PYRIDOXAL
(VitB6)
METABOLISM
3.1.3.74
XENOBIOTIC:
Chemical
pyridoxine
(Vitamin B6)
3.5.4.6
4.3.2.2
AMP
nucleotidase
adenylosuccinate
6.3.4.4
2',3'-cAMP3'-AMP
3.1.4.17
5.3.3.-
RNA
4.6.1.1
cAMP
2.7.4.3ADP
nucleotidase
1.17.4.1
2.7.4.3
dADP
2.4.2.1
3.5.4.4adenosine
2.4.2.1
hypoxanthine
adenine
DNA
2'-deoxyadenosine
2.4.2.1
3.5.4.4
2.4.2.7
2'-deoxyinosine
3.1.3.5dAMP
12-HETE
2.4.2.1
1.2.1.5
methylimidazole
acetaldehyde 1-methylhistamine1.4.3.4
Imidazole-
4-acetaldehyde1.2.1.3
Imidazoleacetic
acid
methylimidazoleacetic
acid
3.4.13.20 3.4.13.186.3.2.11
beta-alanine beta-alaninebeta-alanine
4.1.1.28
2.6.1.58
4.1.1.22
2.1.1.-
histidine
4.1.1.975-hydroxyisourate
5-hydroxy-2-oxo-
4-ureido-2,5-dihydro-1H
imidazole
carnosine
3.5.2.17
5.3.3.-
3.5.3.4
allantoic acid2.4.2.1
allantoin
ureidoglycolateurea
2'-deoxyguanosine
2.7.4.6 dGTP
3.1.3.5
DNA
2.7.7.7
taurine
dGDP
2.7.4.8
2.4.2.8
dGMP
1.4.3.22
2.1.1.8
1.17.3.2
1.17.1.4
1.17.3.2
inosine guanosine
1.7.3.3
2.4.2.1
3.1.3.5
guanine
histamine
1.17.1.4
HISTIDINE
METABOLISM
3.5.4.3
PURINE
METABOLISM
XMPIMP 6.3.5.2 GMP1.1.1.205
nucleotidase
urate
2.4.2.1
xanthine
xanthosine
6.3.4.135-phosphoribosylamine
2.4.2.14
PRPP
orotate
glutamine
2.7.7.83.5.2.36.3.5.5 1.3.5.22.1.3.2
N-carbamoyl-
aspartatecarbamoylphosphate 4,5-dihydroorotate
6.3.3.1
5'-phosphoribosyl-N-formylglycinamidine(FGAM)
glutamine
2.1.2.2
5'-phosphoribosyl-
glycineamide
(GAR)
5'-phosphoribosyl-N-
formylglycinamide
(FGAR)glutamate6.3.5.3
glutamine
3.6.1.17
P1,P4-Bis(5'-uridyl)
tetraphosphate
2.4.2.10orotidylate
3.6.1.19
4.1.1.23
1.17.4.1
UDP
CDP
dCDP2.7.4.6
2.7.4.6
3.6.1.5
3.6.1.5
2.7.4.10
2.7.4.6
3.5.4.5
3.1.3.5
5'-CMP
2.7.1.48
2.4.2.9
dCMP
2.7.1.74
cytidine
2.7.4.14
3.6.1.6
4.1.1.21
UMP3.1.3.5
2.7.1.48
2.4.2.3 uridine
1.3.1.2
5'phosphoriboxyl-5-aminoimidazole
(AIR)uracil
2.7.7.6
dCTP
2.7.7.8
CTP
6.3.4.2
2.7.7.6 RNAUTP
2.7.4.14
3.6.1.5
2.7.4.14
3.6.1.5
RIBOFLAVIN
METABOLISM
2.7.1.158
inositol
1,3,4,6-tetra
kisphosphate2.7.1.140
inositol
1,3,4,5,6-
pentakisphosphate
inositol
hexakisphosphate
FADH2
galactarate
(mucic acid)
glucurono-
6,3-lactone
4.2.1.40
galactarate
(mucic acid)
1.2.1.3
4.2.1.42
5-dehydro-4-deoxy
-D-glucarate
gulonate
1.1.1.19
3.1.1.17
glucuronate
2.7.1.43
3.1.1.191.13.99.1
scyllo-inositol3.1.3.25
3.1.3.57
inositol
1,4-bisphosphate
ISO
inositol 4-P
Inositol
1,3,4-trisphosphate
3.1.3.56
myo-inositolglucuronate
1-phosphate2.4.1.17
2.7.7.442.7.7.64
ASCORBATE
METABOLISM
glucuronate
1.5.1.30
flavin
mononucleotide
3.1.3.2
2.7.7.2
riboflavin
(Vitamin B2)FAD
3.6.1.9
2.7.6.1
2.7.1.26
ribitol
3.1.1.25
ROS
dehydroascorbate
1.1.3.8
ROS
gulono-1,4-lactone
threonate
ascorbate
(Vitamin C)
oxalate
(ethanedioate)
inositol-1,4,5,6-
tetrakisphosphate
2.7.1.153
2.7.1.159
3.1.3.25 4.1.2.20
1.8.5.1
2.7.1.134
glutathione,
oxidized
(GSSG)
glutathione,
reduced
(GSH)
inositol
3,4,5,6-
tetrakisphosphate2.7.1.151 pyruvate
5-hydroxymethylcytosine
5-methylcytosine
5.3.3.-
2'-deoxycytidine
3'-monophosphate
3-methylcytidine
uridine-3'-monophosphate
2',3'-cCMP
3.1.3.66
2',3'-cUMP
pseudouridineDNA
5.3.3.-
5-methyl-2'-deoxycytidine
2.7.7.7
dUTP
3.6.1.19
3.6.1.23
dUDP2.7.4.9
2.7.4.6
2.1.1.45
3.1.3.5
2.7.1.21dUMP
3.5.4.12
2.7.4.9
3.5.1.6
N4-acetylcytidine
ureidoisobutyrate
2.7.4.6
3-aminoisobutanoate
3.1.3.5
2.7.1.21
3.5.2.21.3.1.2
dTMPthymidine
dihydrothymine
dTDP
UDP
1.17.4.1
3.6.1.53.6.1.5
2.7.7.7
dTTP
3-mercaptopyruvic
acid
1.1.1.27
4.4.1.1
thiosulfate2.8.1.2
ROS
2.6.1.1
cystine
2',3'-cGMP
2.6.1.13-sulfinylpyruvate
sulfur dioxide
thiocysteine
SPON
5.3.3.-
pyruvate
3.1.3.36
4.6.1.13
2.7.4.68
2.7.1.673.1.3.B4
3.1.4.11
phosphatidylinositol-4,5-
diphosphate
PI(4)P
3.1.4.3
3.1.3.67
2.7.1.154
PI(3,4)P2
2.7.1.1373.1.3.64
2.7.1.154
Phosphatidylinositol
PI(3)P
3.1.3.86
PI(3,4,
2.7.1.151
2.7.1.150
3.1.3.56
inositol
1,3-bisphosphate
3.1.3.64
1,2-dipalmitoylglycerol
inositol-1,4,5-
triphosphate
2.7.1.159
inositol
2-monophosphate
2.7.1.127
3.1.3.62
3.1.3.25
INOSITOL
METABOLISM
inositol-1,3,4,5-
tetraphosphate
phosphatidylinositol-3,5-
diphosphate,
PI(3,5)P2
2.7.1.64
methylglyoxal
1.1.1.21
monamine
oxidase /
primary amine
oxidase SPON
KETONE
BODIES
aminoacetone
4.1.1.4
1.14.13.n7
1.1.1.103
1.14.99.23
3-hydroxy
benzoate
2,3-dihydroxybenzoate
3.5.1.32
benzoate
1.14.99.15
1-pyrroline-
3-hydroxy-
5-carboxylate
1.5.5.2
4-methoxybenzoate
5.3.3.-
hippurate
1.2.1.-
tetrahydrofolate
2.6.1.23
glycolate
(hydroxyacetate)
4-hydroxyglutamate
semialdehyde
5,10-methylenetetrahydrofolate
2.1.2.1
1.5.8.3
2.1.1.20
2.3.1.29
glycine
2-amino-3-oxobutanoate
sarcosine
(N-methylglycine)
1.2.1.8
choline
betaine
aldehyde
1.1.99.1
threonine
1.5.1.3
folate (Vit B9)
1.5.1.3
7,8-dihydrofolate
dimethylglycine
4.1.1.63
3,4-dihydroxybenzoate
4.1.1.46
catechol
betaine
1.5.8.4
1.14.13.12
GLYCINE
SERINE
THREONINE
METABOLISM
1.14.13.23
1.14.13.334-hydroxybenzoate
3.1.3.77
1,2-dihydroxy-3-keto-
5-methylthiopentene
1.13.11.54
2-oxo-4-methylthiobutanoic
acid
2.6.1.5
1.13.11.53
3.5.1.31
N-formyl-L-methionine
2.1.1.5
1.8.4.11
Detox
BENZOATE
METABOLISM
ROS
methionine
sulfoxide
catechol
sulfate
homocysteine
2.1.1.-
2.5.1.6
3.3.1.1
methionine
S-adenosylmethionine
(SAM)
S-adenosylhomocysteine
(SAH)
2.1.1.13
4.2.1.109
tetrahydrofolate
tetrahydrofolate
1.8.4.1
homocystine
5-MeTHF
5-methylthioadenosine
(MTA)
2.4.2.28
3-mercaptolactic
acid
2.7.1.100
2,3-diketo-5-methyl-
thiopentyl-1-phosphate
3-methylthiopropionic
acid
5.3.1.23
5-methylthioribulose
1-phosphate
5-methylthioribose
1-phosphate
5-methylthioribose
6.3.2.3
ROSgamma-glutamyl-
cysteine
glutathione,
reduced
(GSH)
6.3.2.2
glycine
glutamate
6.3.2.2
gamma-glutamyl-
2-aminobutyrate
6.3.2.3
2-aminobutyrate
5-oxoproline
2.3.2.2
3.4.13.18
cysteinylglycine
Gamma-glutamyl
Amino Acid
cysteine
2.3.2.4
3.5.2.9
cysteine-glutathione
disulfide
ROS
3.1.2.6
lactate
1.11.1.9
glutathione,
oxidized
(GSSG)
GLUTATHIONE
METABOLISM1.8.1.7
2.5.1.18
cis-urocanate
ophthalmate S-methylglutathione
cysteine
sulfinic acidhypotaurine
2.4.2.281.13.11.201.8.1.3 4.1.1.29
6.3.2.2
norophthalmate
6.3.2.3
4.3.1.3
gamma-glutamyl-
alanine
alanine
METHIONINE
CYSTEINE
METABOLISM
methylglyoxal
S-lactoylglutathione
4.4.1.5
Amino acid
thromboxane
A2
6-keto
prostaglandin
F1alpha
prostaglandin
H2
6-keto
prostaglandin
E1
5.3.99.4
5.3.3.-
5.3.99.5
prostaglandin
I2
1.13.11.31
CYP4F/CYP4A
12-HPETE
1.13.11.33
1.14.14.1
1.14.99.1
1.11.1.9 /// ?
8-HPETE
1.13.11.40
11-HPETE
1.13.11.33
11-HETE
1.11.1.9 /// ?
1.11.1.12
8-HETE
1.11.1.9
alpha-KGDH
isocitrate
IDH
2.3.3.14
homocitrate
2.3.1.7
IDH*
alpha-ketoglutarate
succinyl CoA
2-hydroxyglutarate
1.1.1.27
1.4.3.2
2.6.1.42
2-aminobutyrate
2.6.1.1
2-hydroxybutyrate
(AHB)
1.14.13.n7
acetone
1.1.1.21 acetol
1.2.1.24
succinate
semialdehyde
GABA
SHUNT
2.6.1.29
NAAGS
succinylcarnitine
gamma-aminobutyrate
(GABA)
N-acetylaspartate
(NAA)
SPON
N(1)-acetylspermine
creatine
phosphate
Microbiome
creatinine
1.2.1.3
4-acetamidobutanal
putrescine
1.4.3.4
2.3.1.57
N-acetylputrescine
(S)-methylmalonyl-CoA
3.4.17.21
glutamate-5-P
1.2.1.88
2.7.2.11
1.2.1.41L-glutamate
gamma-semialdehyde
2.3.1.57 spermidine
2.5.1.16
1.5.3.162.5.1.22
gamma-glutamyl
Amino Acid
1.14.13.30
1.1.1.-
2.3.2.21.14.99.11.14.99.1
5-HETE
leukotriene C49-HETE sulfate
20-HETE
Amino acid
6.3.2.11
6.3.2.11
anserine
homocarnosine3.4.13.20
1-methylhistidine
imidazole
pyruvate
1.1.1.27
imidazole
lactate
20-OH-prostaglandin
G2
leukotriene
d(,4)
20-hydroxy-
leukotriene-b(,4)20-OH-prostaglandinH2 5-oxoETE 2.3.2.-
3.3.2.-
1.13.11.34
lipoxin B41.13.11.33 CREATINE
METABOLISM
5,6-epoxytetraene
1.8.1.4
arginine2.1.4.1
leukotriene B4
leukotriene
e(,4)
nitric oxide
glycine
1.14.13.342.3.2.-
ornithine
citrulline
EICOSANOIDS
4.4.1.20
leukotriene A4
3.3.2.61.11.1.9
1.13.11.345-HPETE
1.14.14.1
CYP4F/CYP4A
SULF
9-HETE
leukotriene F4
lipoxin A4
20-hydroxy-leukotriene
E4
3.3.2.-
2.3.1.6 3.1.1.7 4.3.1.19
4.2.1.22
1,2-propanediol
acetylcholine
4.4.1.1
cystathionine
alpha-ketobutyrate
4.2.1.49
hydantoin-5
-propionate
3.5.2.7
1.14.13.-
UV
imidazole
propionate
trans-urocanate ?
N-carbamyl-
glutamate
2.1.2.5
formimino-
glutamate
Microbiome
5.3.3.-
argininosuccinate
6.3.5.4
5.3.3.-
1.14.13.39
fumarate
N-acetylarginine
6.3.4.54.3.2.1
asparagine
2.1.3.3
1.4.1.3CO2propionyl CoA
NH3
aspartate
2.3.1.17
4.1.1.15
6.4.1.3
3.5.1.15
6.3.4.16
6.3.1.2
carbamoyl-phosphate3.5.1.2
glutamateglutamine
agmatine
3.5.3.1
2.1.1.2
ARGININE
METABOLISM
AND
UREA
CYCLE
4.1.1.19
urea
guanidinoacetate
ornithine
POLYAMINE
METABOLISM2.7.3.2
2.6.1.13
3.5.3.11
4.1.1.17
creatine
prostaglandin
D2
11beta-prostaglandin
F2alpha
delta12-prostaglandin
J2
1.1.1.188
5.3.3.-
5.3.99.2
prostaglandin
J2
5.3.3.-
PGG2
1.14.99.11.1.1.184
1.14.99.1
5.3.99.3
15-HPETEprostaglandin
E2
arachidonate
(20:4n6)
1.1.1.188
15-keto
prostaglandin
F2alpha
1.1.1.196
1.1.1.188
prostaglandin
F2alpha 1.1.1.189
1.3.1.48
1.1.1.141
1.1.1.189
15-keto
prostaglandin
E2
5,6-EET 8,9-EET 11H-14,15-EETA15H-11,12-EETA11,12-EET 14,15-EET 15-HETE16(R)-HETE
3.3.2.10 1.14.14.1 1.14.14.11.3.1.48 3.3.2.103.3.2.103.3.2.10 1.1.1.232
1.14.14.1 1.14.14.11.14.14.1 1.14.14.11.14.14.11.14.14.1 1.11.1.9 1.14.14.1
20:3n9
sphingosyl
phosphorylcholine
3.1.4.12
2.7.8.27
SPHINGOLIPID
METABOLISM
3.5.1.23
2.3.1.24
sphingosine
Phosphatidylserine
(PS)
Phosphatidylinositol
(PI)
12/15 LOX
ROS RNS
Phosphatidylethanolamine
(PE)
1.1.1.102
3-ketosphinganine
Acyl-SN-
glycerol-3P
2.3.1.51
L-Phosphatidate
1.14.-.-
2.3.1.15dihydroceramide
1.14.-.-
sphinganine
2.7.7.41
glycerophosphoethanolamine
CDP-diacylglycerol2.7.8.11glycerophosphorylcholine
(GPC)
3.1.1.5
Oxidized
Phospholipids
2.7.1.30
Lysolipids
PLA
glycerol
3-phosphate
(G3P)
2.7.8.-
3.1.3.4
Diacylglycerol
(DAG)
PE-N-methyltransferases
2.7.8.29
Phosphatidylcholine
(PC)Long Chain
Acyl-CoA /
Long Chain
Fatty Acids
2.3.1.50
serine
N-acetylproline5.3.3.-
1.5.1.2
PROLINE
METABOLISM1.5.99.8
proline
1.14.19.-
E5
1.14.19.3
20:2n9
18:2n9
lyso-dihydro
sphingomyelin
ethanolamine
nervonate
(24:1n9)
arachidate
(20:0)
E1/E3
behenate
(22:0)
E1/E3
E1/E3
2.3.1.9
lignocerate
(24:0)
E1/E3
3.1.2.2
erucate
(22:1n9)
oleate (18:1n9)
(R)-methylmalonyl-CoA
5.1.99.1
5.4.99.2
ASPARTATE
GLUTAMATE
METABOLISM
N-acetyl-aspartyl-
glutamate
(NAAG)
palmitoleate
(16:1n7)
1.14.19.1
myristoyl-CoA
3.1.2.2
myristoleoyl-coa
3.1.2.2
myristate
(14:0)
2.3.1.85
6.2.1.3
myristoleate
(14:1n5)
E1/E3
palmitoleoyl-CoA
6.2.1.3
palmitoyl-CoA
1.14.19.1
E1/E3
cis-vaccenate
(18:1n7)
palmitate
(16:0)
oleoyl-CoA
1.14.19.1
gondoate
(20:1(n-9))
E1/E3
E1/E3
stearoyl-CoA
3.1.2.26.2.1.3
stearate (18:0)
22:4n6
choline
phosphate
phosphoethanolamine
2.7.1.32
2.7.1.82
choline
3.5.1.99
ENDOCANNABINOID
SYNTHESIS3.1.4.4
Triacylglycerol
(TAG)
Endocannabinoid
SPO3.5.1.63spermine
gamma-aminobutyrate
(GABA)
(S)-1-pyrroline-
5-carboxylate
4-acetamidobutanoate
3.5.1.99
3.1.1.3
C-18 Oxylipids
N-palmitoyl
taurine
5.3.3.-
linoleate
(18:2n6)taurine
3.1.1.4
N-stearoyl
taurine
2.7.7.14
2.3.1.20
cytidine-5'-diphospho
ethanolamine
2.7.7.15
cytidine
5'-diphosphocholine
2.7.8.1
2.7.8.2
15-KETE
11,12,15-
THETA
N-methylhydantoin
11,14,15-THETA
medium chain
3-hydroxy
acyl-CoA
3.1.2.2
4.2.1.17
1.3.8.7
1.1.1.35trans-D2-Enoyl-CoA
15-deoxy
delta-12,14-prostaglandin
J2
13,14-dihydro-15-
keto-prostaglandin
E213,14-dihydro-15-keto
-prostaglandin
F2alpha palmitate
(16:0)
14,15-DHET5,5-DHET
5.3.3.-
8,9-DHET 11,12-DHET
oleate (18:1n9)
4.2.1.17
short chain
3-hydroxy
acyl-coA1.3.8.1
Medium-chain
3-hydroxy
fatty acid
sc trans d2
enoyl-CoA
2.3.1.137
3-ketoacyl-CoA
2.3.1.13
2.3.1.16
Short-chain
acyl-CoA
13,14-dihydroprostaglandin
F2alpha N-arachidonoyldopamine
1.1.1.189
N-oleoyldopamine
3.5.1.99
N-palmitoyldopamine
20:3n6
1.1.1.35
acetoacetyl
CoA
20:5n3
22:6n3
3.1.2.2
Short-chain
acylcarnitine
Long Chain
Fatty Acid
Short-chain
acylglycine
Medium Chain
Fatty Acid
6.2.1.2Medium-chain
acylcarnitine
3-ketoacyl-CoA
2.3.1.137
2.3.1.16
1.1.1.211
Medium-chain
acyl-CoA
L-3-HYDROXYACYL-COA
coenzyme A
Long-chain
acyl-CoA
PHOSPHOLIPID
METABOLISM
6.2.1.3
Long-chain
3-hydroxy
fatty acid
DICARBOXYLIC
ACID
METABOLISM
3.1.2.2
6.2.1.3
6.2.1.3
fatty omega
aldo acid
1.2.1.3
MC
dicarboxylate
acyl-CoA
1.1.1.1
1.3.8.7
fatty omega
hydroxy acid
Long-chain
dicarboxylate
fatty acid
Medium-chain
dicarboxylate
fatty acid2.3.1.16
adipate
6.2.1.3
succinate
BETA-OXIDATION
4.2.1.17
1.3.8.8TRANS-D2-
ENOYL-COA
1.3.8.9
1.1.1.35
4.2.1.17
Medium-chain
3-hydroxy
dicarboxylate
fatty acid
3.1.2.2
MC 3-hydroxy
dicarboxylate
acyl-CoA
3-ketoacyl-CoA
trans-D2-
Enoyl-CoA
1.3.1.3
5.3.3.-
21-hydroxy-5b-
pregnane-3,11,20-trione
11-dehydro
corticosterone
11b,21-dihydroxy-3,20-
oxo-5b-pregnan-18-al aldosterone
1.1.1.50
3a,21-dihydroxy-
5b-pregnane-11,20-dione
beta-ox
N,N-dimethyl
sphingosine
1.14.19.3
24:6n3
3a,11b,21-trihydroxy-
20-oxo-5b-pregnan-18-al
22:5n6 24:5n6
1.1.1.50
22:6n3
3.5.1.109
5.3.3.-
fatty acid5.3.3.-
erythro-sphingosyl
phosphorylcholine
galactosylsphingosine
Sphingomyelins
3.1.3.4
5.3.3.-
2.7.1.91
2.4.1.23
erythro-sphingosine-
1-phosphate
1.3.8.8
trans-d2-Enoyl-coA
LC
dicarboxylate
acyl-CoA
1.14.15.3
2.8.2.4
1.14.14.1
1.14.14.1
estrone sulfate
glycerol
estrone
1.14.14.1
1.14.14.1
2.4.1.17
estrone
glucuronide 1.1.1.146
cytidine-5'-
diphosphoglycerol2.7.7.39
11b-hydroxyandrost-
4-ene-3,17-dione
1.14.15.4
1.14.14.1
19-oxoandrost-
4-ene-3,17-dione
2.4.1.17
2-methoxyestrone
3-glucuronide
2-methoxyestrone
16a-hydroxyestrone
2.1.1.62-hydroxyestrone
2.4.1.171.14.99.11
3-ketoacyl-CoA
2.8.2.15
2.4.1.17
2-methoxy-estradiol-17b
3-glucuronide
estradiol-17beta
3-glucuronide
2.3.1.166beta-hydroxy
estradiol-17beta
beta-estradiol
3-sulfate
1.14.14.12-hydroxyestradiol
1.14.14.1
estradiol
1.1.1.211 2.1.1.6
2-methoxyestradiol
LC 3-hydroxy
dicarboxylate
acyl-CoA2.4.1.17
estriol
1.1.1.62
1.1.1.62
16-glucuronide-estriol
4.2.1.17
3.5.1.232.3.1.24
Ceramides
phytosphingosine
L-1-phosphatidyl-
glycerol3.1.3.27
2.7.8.5
L-1-phosphatidyl-
glycerol-P
2.3.1.212.3.1.21
Long-chain
acylcarnitine
Long-chain
acyl-CoA
2.7.8.-
3.1.4.2
Cardiolipin
3.1.4.2
tetrahydrocortisol
1.3.1.3
1.1.1.50
11b,21-dihydroxy-
5b-pregnane-3,20-dione
11beta,17alpha,21-trihydroxy-
5beta-pregnane-3,20-
4.1.2.30
21-deoxycortisol
allopregnanolone
5alpha-pregnan-
20alpha-ol,3-one
1.14.99.10
corticosterone
cortisol
1.14.15.5
1.1.1.-
18-hydroxy
corticosterone1.14.15.4
1.1.1.50
1.1.1.146
1.1.1.146
1.1.1.-
1.3.1.3
5.3.3.-
4.1.2.30
cortisone
20:3n620:4n3
18:3n6
E5
1.14.19.-
1.14.19.3
18:2n618:3n3
18:n3
24:5n3
24:4n6
FATTY
ACID
METABOLISM
E2/E5
22:5n3
arachidonate
(20:4n6)
Plasmalogen
Fatty Acids
E2/E5
alkenyl-GPC-hydrolase
/ plasmalogen
synthase
dihydroxyacetone
phosphate
(DHAP)
20:5n3
3-b-HSD / SDI
1.1.1.50
testosterone
glucuronide
1.14.14.1
dihydrotestosterone
7a-hydroxytestosterone
16a-hydroxydehydro
isoandrosterone
5a-androstane-3,17-dione
16a-hydroxyandrost-
4-ene-3,17-dione
1.3.1.22
androsterone
1.1.1.50
3.1.6.2
dehydro
epiandrosterone
1.14.13.17
2.8.2.2
tetrahydrocorticosterone
17a,21-dihydroxy-
5b-pregnane-3,11,20-trione
3-b-HSD / SDI
1.14.14.1
7-alpha-hydroxycholesterol
7a-hydroxyandrost-
4-ene-3,17-dione
tetrahydrocortisone
1.1.1.146
1.1.1.50
5beta-androstan-3alpha,17beta-diol
DHEA sulfate
testosterone
1.1.1.641.1.1.239
androstenedione
1.3.1.3 2.4.1.17
1.14.14.1
1.3.99.5
19-hydroxyandrost-
4-ene-3,17-dione
19-hydroxyandrost-
4-ene-3,17-dione
1.14.14.1
1.14.14.1
19-oxotestosterone
1.14.14.1
19-hydroxytestosterone 1.14.14.1
5b-dihydrotestosterone
adrenosteroneetiocholanolone
2.4.1.17
etiocholanolone
glucuronide
androsterone
glucuronide
2.4.1.17
etiocholanedione
1.3.1.3 1.1.1.50
Copyright 2015 Metabolon, Inc.
Normal metabolomics result
Circle size relative to Z-score
High Low -1.5 - +1.5 Molecule in library but not detected Molecule not in library
Global MAPS Global Metabolomic Assisted Pathway Screen
• Metabolic pathway screen for perturbations in levels of analytes and relative abundance – Screens for >2500 small molecules (50-1500 Da)
– Z-scores provided (not absolute values)
• Small molecule metabolomic analysis – Plasma
• ~750-900 analyte identifications per plasma sample
– CSF • ~300 analyte identifications per CSF sample
– Urine • ~1200 analyte identifications per urine sample
Limitations of test 1. Inappropriate for identification of
• Analytes >1500 Da or <50 Da • Proteins/large peptides • Complex oligosaccharides • Large lipids • Elements (K, Na, etc.)
2. Analytes requiring special extraction/chromatographic separation
• Homocysteine (requires reductant treatment)
3. Screening tool to identify metabolic perturbations
4. Values are not quantitative
5. Not for acute assessments
If you are interested in a specific compound we can provide information on detection rates, accuracy, and analyte stability.
Sample collection/validation
•Retrospectively collected from stored lab
samples
•Na-Heparin treated plasma, stored -20 C for up
to 3 months
•83% from Texas Children’s Hospital
•“Normal Controls”
•Patient that came to our lab for testing but for
whom no abnormal analytes were detected
•Roughly age and sex matched to known patient
samples
Overview of Plasma Samples
•200 total •128 from patients with
diagnosis of IEM
•72 “Normal Controls”
•27 different IEMs
•Majority of patients
on treatment
Overview of samples
•200 total •128 from patients with
diagnosis of IEM
•72 “Normal Controls”
•27 different IEMs
•Majority of patients
on treatment
0 10 20 30 40 50 60 70 80
unknown
Urea cycle
organic acidemia
branch chain aa
other
creatine
cofactor…
fatty acid…
# of samples
Current analyte detections possible in our lab
All biochem lab plasma tests
Average metabolomic detection in plasma
Average metabolomic detection in plasma
Clinical validation experiments
r = 0.94
•Intra-assay precision •median=10.47% (IQR= 5.55-
22.04%)
•Linear detection •34 acylcarnitines and amino
acids
•median r=0.9, IQR r= 0.84-0.95
•Stability •Plasma separation time course
studies- 30 mins to >1 day
Expected IEM-related analyte elevations were detected
Expected IEM-related analyte elevations were detected
PKU
Methylmalonic acidemia
Methylmalonic acidemia
Metabolomic Results: Argininemia
Plasma metabolomic analysis successfully screened for 20 different IEMs
• Urea cycle disorders
– Arginase def – Argininosuccinate lyase def – Citrullinemia – Ornithine transcarbamylase def
• Amino acid disorders – Homocystinuria (CBS) – Maple syrup urine disease – Phenylketonuria
• Fatty acid oxidation disorders – MCAD – VLCAD
• Organic acidemias – 3-methylcrotonyl-CoA
carboxylase def – Cobalamin disorders – Glutaric acidemia type I – HMG-CoA lyase def – Isovaleric acidemia – Methymalonic acidemia – Propionic acidemia
• Other – Guanidinoacetate
methyltransferase def – Holocarboxylase synthetase def – Thymidine phosphorylase def – TMLHE def
results
FUNCTIONAL VALIDATION OF DNA VARIANTS OF UNCERTAIN SIGNIFICANCE
Neurologic phenotypes
Case 1 – Citrate Transporter Deficiency EXOME Findings
• trans mutations in SLC13A5
•c.997C>T (p.R333X) and
c.680C>T (p.T227M)
•Disorder: AR citrate transporter def.
TCA
cycle
Citrate
Case 2 - Aromatic Amino Acid Decarboxylase Deficiency
• 4 year old infant with developmental delay and hypotonia; initial presentation 11 months
• Tests performed previously– VLCFA, LSD panel, urine MPS, CMA,PAA, UOA, ACP, NH3, lactate, CK, CSF glucose/protein, muscle biopsy, ETC analysis, mitochondrial genome/depletion, MRI brain
• WES – 2 VUS (trans), c.286G>A (p.G96R) and c.260C>T (p.P87L) in the DDC gene
Case 2 - Pathway and Results
Patient is shown in red
Z sc
ore
VMA
Baylor Genetics Circle size relative to Z-score
High Low -1.5 - +1.5 Molecule in library but not detected Molecule not in library
Aromatic amino acid decarboxylase deficiency
Dopa-containing medications
Case 3
• 19 month old male – Global developmental
delay
– Hypotonia
– Abnormal movements
– Abnormal MRI (delayed myelination)
– Oculomotor apraxia
– Facial hemangioma
– Constipation
• Prior normal workup – Microarray
– Metabolic workup
• Plasma amino acids
• Lactate
• Ammonia
• Urine organic acids
• CSF amino acids
• CSF neurotransmitter profile
Whole exome sequencing and metabolomics ordered
Case 3 WES Results
• Single heterozygous pathogenic variant – UROC1, novel c.1448_1449delCT (p.S483fs)
• Urocanase deficiency [MIM #276880 ]
• Two heterozygous VUS – ABAT, novel: c.454C>T (p.P152S) and c.1393G>C (p.G465R)
• GABA transaminase deficiency [MIM #613163 ]
Single heterozygous VUS • ACAD9, ACAD9 deficiency • ATM, Ataxia-telangiectasia • UPB1, Beta-ureidopropionase deficiency • DARS, Hypomyelination with brainstem and spinal cord involvement and leg spasticity • CSPP1, Joubert syndrome 21 • HERC2, Mental retardation, autosomal recessive 38 • TH, Segawa syndrome, recessive • SPG11, Spastic paraplegia 11, autosomal recessive • AP4B1, Spastic paraplegia 47, autosomal recessive
Case 3 WES Results
• Single heterozygous pathogenic variant –UROC1, novel c.1448_1449delCT (p.S483fs)
•Confirmed by Sanger sequencing: Coverage = 100%
»Father heterozygous
»Mother negative
•Urocanase deficiency [MIM #276880 ]
• Two heterozygous VUS
– ABAT, novel: c.454C>T (p.P152S) and c.1393G>C (p.G465R)
• GABA transaminase deficiency [MIM #613163 ]
Case 3 - Metabolomic Results
Histidase
Histidine Urocanic acid
Imidazole propionic acid
Urocanase
Imidazolone propionic acid
UROC1
Cis- and trans-urocanic elevations
No significant alterations of molecules in histidine pathway.
Second pathogenic variant likely not present.
Diagnosis likely not urocanase deficiency.
Case 3 WES Results
• Single heterozygous pathogenic variant –UROC1, novel c.1448_1449delCT (p.S483fs)
•Confirmed by Sanger sequencing: Coverage = 100%
»Father heterozygous
»Mother negative
•Urocanase deficiency [MIM #276880 ]
• Two heterozygous VUS
– ABAT, novel: c.454C>T (p.P152S) and c.1393G>C (p.G465R) •c.454C>T (p.P152S), novel, inherited from mother
•c.1393G>C (p.G465R), novel, inherited from father
•Both variants predicted to be deleterious using sift and polyphen
•GABA transaminase deficiency [MIM:#613163]
Plasma (n=124)
2-pyrrolidinone
Patient samples Plasma CSF
-4-2
02
466
4
2
0
-2
-4
Plasma CSF
-4-2
02
46
Z sc
ore
2-pyrrolidinone - a new biomarker for ABAT deficiency
GABA transaminase (ABAT) deficiency (Plasma!)
2-pyrrolidinone
Glutamate
GABA
Succinic semialdehyde
Glutamic acid decarboxylase
GABA-transaminase
Succinate
2-oxoglutarate
Exome + Metabolomics
• 180 Cases with both clinical exome and clinical metabolomic testing
• Assessed diagnostic rate of platforms
• Assessed when metabolomics contributed to variant re-classification [Alaimo, ASHG 2017]
Contribution of Metabolomics
Provided Information
Did not aid in the case
62.2% 37.8%
Contribution of Metabolomics to Genomics
Diagnosis
Molecularly Solved
50.5% 49.5%
Contribution of Metabolomics
Provided Information
Did not aid in the case
75%
14.7%
19.1% Areas Improved
Rules Out Variants fromDiagnosis
Variant Classificaiton Change
Confirms Molecular Diagnosis
62.2% 37.8%
Contribution of Metabolomics to Genomics
75%
14.7%
19.1%
Areas Improved
Rules Out Variant fromDiagnosis
Variant Classification Change
Confirms Molecular Diagnosis
Contribution of Metabolomics to Variant Interpretation
Variant Classification Changes B LB VUS LP P
6
5
2
1
1
B = benign LB = likely benign VUS = variant of uncertain significance LP = likely pathogenic P = pathogenic
FURTHER VALIDATION AND NEW DISCOVERIES
Peroxisomal Biogenesis disorders (PBD)
Classic Zellweger Neonatal Adrenoleukodystrophy Infantile Refsum
Disease Phenotype
PEX alleles
Severe hypomorphic or null Mild hypomorphic
Biochemical Phenotype
↑ ↑ VLCFA’s
Absent or deficient peroxisomes ↑ or normal VLCFA’s
Reduced # peroxisomes
Pictures from Inborn Metabolic Diseases 4th edition and SIMD NAMA
PBD are a clinical spectrum of disease
Disease Severity
Mild PBD
• 7 year old • Phenotype mimicking
Usher syndrome with hearing loss and pigmentary retinopathy, normal cognition, diagnosed by research sequencing study for Usher
• PEX1 G843D homozygote
PBD
Controls
Long-chain Dicarboxylic Acids
Phosphatidylcholines (PC)
Phosphatidylethanolamines (PE)
Plasmalogen phospholipids
Sphingolipids
Relative increase
Relative decrease Amino Acids
Peptides
Carbohydrates
Energy
Lipids
Nucleotides
Cofactors
Xenobiotics
Zellweger-spectrum disorders Metabolomics
~650 named molecules identified in each plasma sample, N=19
Component 1 [18.98%]
-10 0 10 20 -20 -30 -40
-50
-40
-30
-20
Co
mp
on
en
t 2 [
10.1
0%
]
-10
0
10
20
C24 (VLCFA)
1-(1-enyl-palmitoyl)-GPC*
1-(1-enyl-stearoyl)-GPC*
Hexadecanedioate
Eicosanodioate
-4 6 4 0 2 -2 -6
Pipecolic Acid
7-HOCA (bile acid)
Octadecanedioate
Docosadioate
1-(1-enyl-oleoyl)-GPC*
1-O-hexadecyl-GPC*
Z Score *Plasmalogens
Results: Untargeted metabolomic analysis
Results: Sphingomyelins as new biomarkers for PBD-ZSD
Palmitoleoyl Sphingomyelin
Behenoyl Sphingomyelin*
Nervonoyl Sphingomyelin
Euricoyl Sphingomyelin
Palmitoyl Sphingomyelin
Oleoyl Sphingomyelin
Myristoyl Sphingomyelin
Eicosenoyl Sphingomyelin
Sphingomyelin
-4 6 4 0 2 -2 -6 Z Score
NEW DISCOVERIES!
Oral Carnitine is converted to Trimethylamine N-oxide (TMAO)
Backhed F., Nature Medicine (2013) PMID: 23652100
TMAO
Carnitine
Gut Microbiota
Oral Carnitine is converted to Trimethylamine N-oxide (TMAO)
Backhed F., Nature Medicine (2013) PMID: 23652100
Carnitine
Gut Microbiota Koeth R.A., et al., Nature Medicine (2013) PMID:23563705
Koeth R.A., et al., Nature Medicine (2013) PMID: 23563705
Diet & baseline production of TMAO
Dietary influence of carnitine-challenge on TMAO production
Koeth R.A., et al., Nature Medicine (2013) PMID: 23563705
Plasma TMAO elevations in Global MAPS validation cohort
Plasma TMAO elevations in Global MAPS validation cohort
Meat restriction and oral carnitine supplementation
•Clinical notes on 19 of 24 patients •All on Chronic PO carnitine (range 17-145 mg/kg/day) •All highly discouraged from consuming meat
Meat restriction and oral carnitine supplementation
•Clinical notes on 19 of 24 patients •All on Chronic PO carnitine (range 17-145 mg/kg/day) •All highly discouraged from consuming meat
Disorder 3-methylhistidine (average z-score) p value
organic acidemia -0.70 5.02E-04
urea cycle -0.73 1.82E-05
pku -0.57 7.96E-03
no diagnosis 0.00 NA
Plasma Carnitine is decreased in patients with organic acidemias
Plasma Carnitine is decreased in patients with organic acidemias
TEST REPORTING
Reporting Format
• Analysis of data
• Interpretation provided by laboratory director
• Tables of analytes with Z-score >+2 or <-2
– Human metabolites
– Drugs
– Xenobiotics
– Dietary
• Analytes categorized by pathway
Summary Global MAPS • Identifies all common IEMs studied to date screened on
PAA/UOA/ACP
• Screening tool for undifferentiated phenotypes – Developmental Delay/Intellectual disability/Hypotonia
– Seizures (non-specific)
• Does not replace PAA, UOA, etc. for diagnostic testing or management nor can it detect large molecules (MPS, CDG)
• Validate DNA results and can identify IEMs for which no biochemical testing available (Citrate transporter deficiency)
• Potential to diagnose neurotransmitter disorders on a plasma specimen (AADC & GABA transaminase)
• Discovery of Novel Biomarkers for IEMs (PBDs)
• Understand effects of therapies in IEMs
Taraka Donti Marcus Miller Paldeep Atwal
Bret Bostwick
Sarah Elsea
Qin Sun Leroy Hubert
Adam Kennedy
Michael Wangler
Lisa Emrick
What does Global MAPS test for?
• Disorders of amino acid metabolism: plasma amino acids $220
• Organic acidemias: urine organic acids and
acylglycines $500
• Purine disorders: urine purine panel $260
• Pyrimidine disorders: (urine pyrimidine
panel $280
• Neurotransmitter disorders: plasma
thymidine; urine pyrimidines; plasma/urine
creatine & guanidinoacetate; csf:
succinyladenosine, 5HIAA, HVA, 3OMD, lactate,
& glucose $1330 (exclusive of LP costs)
• Cholesterol Metabolism & PBDs $850
• Creatine disorders: plasma/ urine
creatine and guanidinoacetate $280
• Bile acid disorders: plasma/urine bile
acids $917
• Urea cycle disorders: plasma amino
acids & urine orotic acid $300
• Fatty acid oxidation disorders: acylcarnitine profile, acylglycines, & urine organic acids $770
• Certain mitochondrial disorders: – MNGIE: plasma thymidine $200
Total cost: > $5000!
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