testing untargeted metabolomic profiling in inborn errors...

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

http://upload.wikimedia.org/wikipedia/commons/9/9a/Cholesterol.svg

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


Interpretation


Compound ID


QA/QC

Data Reduction


UHPLC (HILIC)-MS/MS



UHPLC-MS/MS (-ESI)

Enrich 50-1500 Da small molecule analytes


Interpretation


Compound ID


QA/QC

Data Reduction


•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)


Interpretation


Compound ID


QA/QC

Data Reduction


•Library of ~2500 human metabolites

UHPLC (HILIC)-MS/MS



UHPLC-MS/MS (-ESI)


Interpretation


Compound ID


QA/QC

Data Reduction


Pathway Visualization: Metabolync™ plugin to Cytoscape developed to overlay analyte findings onto metabolic pathways

UHPLC (HILIC)-MS/MS



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


1.11.1.8

1.11.1.8

5a-pregnane-3,20-dione


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


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.-


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

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

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

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

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!

testing untargeted metabolomic profiling in inborn errors...

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