testing
Untargeted Metabolomic
Profiling in Inborn Errors ofprevention
Metabolism
research
V. Reid Sutton, MD education Professor, Molecular & Human Genetics
Baylor College of Medicine
Medical Director, Biochemical Genetics
Laboratory 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
Biochemistry DNA Advantages
Any sample type RNA Condensed & information rich
Proteins Actionable
OH O H O O OH
O H HO OH N H OH 2 Biochemicals Bridge between glucose cholesterol threonine genome & phenotype Mechanistic Insight into Phenotype
9 METHODS Metabolon, a global leader in metabolomics 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 UHPLC-MS/MS (+ESI) Early/Polar Library Search Biochemical UHPLC-MS/MS (+ESI) Late/Lipid RT, Mass, MS/MS Extraction Data Reduction UHPLC-MS/MS (-ESI) Compound ID QA/QC UHPLC (HILIC)-MS/MS
Pathway Visualization: Metabolync™ plugin to Cytoscape developed to Statistical Analysis overlay analyte findings onto metabolic pathways
Biomarkers Mechanistic Understanding Drug MoA Interpretation Cellular Characteristics
UHPLC-MS/MS (+ESI) Early/Polar Library Search Biochemical UHPLC-MS/MS (+ESI) Late/Lipid RT, Mass, MS/MS Extraction Data Reduction UHPLC-MS/MS (-ESI) Compound ID QA/QC UHPLC (HILIC)-MS/MS
Enrich 50-1500 Da small molecule analytes Statistical Analysis
Biomarkers Mechanistic Understanding Drug MoA Interpretation Cellular Characteristics
UHPLC-MS/MS (+ESI) Early/Polar Library Search Biochemical UHPLC-MS/MS (+ESI) Late/Lipid RT, Mass, MS/MS Extraction Data Reduction UHPLC-MS/MS (-ESI) Compound ID QA/QC UHPLC (HILIC)-MS/MS
•Q exactive MS/MS - Orbitrap based MS/MS (Thermo) •Accurate to 1 ppm vs 100 ppm for quadrupole analyzers Statistical Analysis •Cost ~2X quadrupole
Biomarkers Mechanistic Understanding Drug MoA Interpretation Cellular Characteristics
UHPLC-MS/MS (+ESI) Early/Polar Library Search Biochemical UHPLC-MS/MS (+ESI) Late/Lipid RT, Mass, MS/MS Extraction Data Reduction UHPLC-MS/MS (-ESI) Compound ID QA/QC UHPLC (HILIC)-MS/MS •Library of ~2500 human metabolites
Statistical Analysis
Biomarkers Mechanistic Understanding Drug MoA Interpretation Cellular Characteristics
UHPLC-MS/MS (+ESI) Early/Polar Library Search Biochemical UHPLC-MS/MS (+ESI) Late/Lipid RT, Mass, MS/MS Extraction Data Reduction UHPLC-MS/MS (-ESI) Compound ID QA/QC UHPLC (HILIC)-MS/MS
Pathway Visualization: Metabolync™ plugin to Cytoscape developed to Statistical Analysis overlay analyte findings onto metabolic pathways
Biomarkers Mechanistic Understanding Drug MoA Interpretation Cellular Characteristics
Metabolon’s Approach to Metabolomics - a Comprehensive Survey of the Metabolome
17
Extensive Metabolite Library
High resolution biochemistry surveys central metabolism & Baylor Genetics peripheral pathways that 18 drive attributes of phenotype.
te a n o r u c u l g
- o r d y h e -d P-4 D G e n i m ta u l g isoleucine leucine 2.4.1.18 - -N l y s o b i r o h p s o h -p ' 5 - l y s o b i r o h p s o h -p ' 5 e s o n n a -m -D y x o e -d 6 2.6.1.42 e n i m ta u l g e n i m a s o c u l -g l ty e c -a N 1 2.4.1.1 e n i c u e l o s i o l l -a L valine e d i m a n i c y l g l y m r fo glycineamide te a h p s o h -p 1 e n i m a s o c u l g l ty e c -a P-N D U te a m ta u l g 2.7.7.23 isovalerylalanine ) R A G (F ) R A (G e n i m a l y s o b i r o h p s o h -p 5 2.7.7.44 5.4.2.3 te a r e l a v o s i y x o r d y -h 3 6.3.5.3 2.1.2.2 6.3.4.13 2.4.2.14 3.1.4.3 2.7.7.64 N IO T A L Y S CO Y L G 2.6.1.42 3.1.3.25 5.3.3.- l to i s o n i te a r e l a v o x -o -2 l y th e -m 3 2.6.1.42 1.4.3.2 l to i s o n i l y d ti a h p s o Ph GDE te a h p s o h p o n o -m 2 e n i m a s o c u l g l ty e c -a N 2.6.1.42 l to i s o n -i o y m e s o c u l P-g D U te a n o c a s e m te a n o r u c u l g 1.1.1.271 4.2.1.47 E T RBA CO S A te a h p s o h -p 6 te a r e l a v o s i y x o r d y -h 4 ) te a r a m fu l y th e (m te a h p s o h -p 1 4.6.1.13 2.7.1.64 2.4.1.17 -1 (n n e g o c y l g - l y th e -m 4 SO I Glycogen PP PR M IS L BO A T E M 1.2.1.- te a o r p a c o s i y x o r d y -h a h p l a ? residue) E T A N E H T O T N PA te a r e l a v l y th e -m -3 y x o r d y -h 2 te a r e l a v o x -o 2 te ta o r o ? te a r e l a v o x -o -2 l y th e -m 3 e s o c P-fu D G e s o n n a P-m D G e n i m a s o c u l g l ty e c -a N te a r ty u b o x -o -2 l y th e -m 3 - l y o m a b r a -c N 2.7.1.59 te a r e l a v o s i y x o r d y -h a h p l a ? M) A G (F e n i d i m a n i c y l g l y m r -fo -N l y s o b i r o h p s o h -p ' 5 l to i s o n -i o l l y c s B5) IT (V 3.1.3.25 - -1 l y th e -m 3 te a h p s o h p l y o m a b r a c te ta r a p s a te ta o r o o r d y h i -d 5 , 4 te a m ta u l g l y r e l a v o s i te a n i c c u s l y th e m e n i m ta u l g - -1 l y th e -m 2 PP h -T l ty u b y x o r d y h nd CoA nd a - -1 l y th e -m 2 3.1.3.64 2.7.1.43 A o -C l y r ty u b l y th e -m )-2 (R PP h -T l ty u b y x o r d y h 2.3.1.4 5.1.3.2 glucurono- N E G CO Y L G M IS L BO A T E M PP h -T l y p o r p y x o r d y h -P 4 l to i s o n i e n to c a -l 3 , 6 2.7.7.9 BCKD - 5 , -3 l to i s o n i l y d ti a h p s o h p GTP BCKD 6.3.5.5 2.1.3.2 3.5.2.3 1.3.5.2 3.1.3.64 2.7.1.137 3.1.3.B4 2.7.1.67 2.7.7.13 2.7.7.22 2.4.1.1 BCKD isovaleryglucuronide isovalerylsarcosine 2.7.7.8 e n i e th te n a p , te a h p s o h p i d M IS L BO A T E M te a n e th to n a p 3.5.1.92 6.3.3.1 l to i s o n i T PG F e s to c a l a P-g D U 1.3.99.- te a n o r u c u l g 2.7.7.12 - S-(2 )P2 5 , (3 PI 1.13.99.1 3.1.1.19 )- l y o n ta u b l y th e -m S-(2 )- l y o n ta u b l y th e -m S-(3 te a h p s o h p s i -b 3 , 1 te a h p s o h -p -6 e n i m a s o c u l g 2.7.1.1 glucosamine 2.4.1.1 n i tr x e d to l a m te a l y d ti o r o 2.4.2.10 glucose ) l y n o i p o r p l y th e dihydrolipoamide -E e d i m a o p i l o r d y h i d ) l y d i r -u ' (5 s i -B P4 , P1 ) (n te a o n ta p e h o s i y x o r d y -h 3 te a h p s o h -p 1 -E e d i m a o p i l o r d y h i -d te a h p s o h -p -1 e s o n n a m te a h p s o h p a tr te 3.1.3.57 2.7.1.33 ROS 3.6.1.19 te a h p s o h -p -1 e s o c fu A o -C l y l y r c a l y th -e 2 te a r ty u b l y th e -m 2 3.1.2.20 A o -C l y r ty u b l y th e -m 2 te a r e l a v o s i e s to c a l a g te a r ty u b o s i A o -C l y r ty u b o s I A o -C l y r e l a v o s i 3.1.2.20 3.1.2.20 5.3.3.- 4.1.1.23 1.1.1.19 te a h p s o h -p 1 e s o a x e h to l a m 2.7.7.10 3.6.1.17 1.2.1.3 2.7.1.48 2.7.4.6 3.5.99.6 2.6.1.16 2.7.4.14 )P (3 PI )P (4 PI 2.7.1.150 l to i s o n i e n i th te n a p e l zo a d i m i o n i m -a -5 l y x o b i r o h p s o h p ' 5 5.4.2.2 5.4.2.8 2.4.1.1 uracil MP U UDP UTP RNA te a h p s o h p s i -b 4 , 1 te a n e th to n a p o h p s o h -p ' 4 4.2.1.17 ) R I (A 2.4.2.3 uridine 3.1.3.5 3.6.1.6 2.7.4.10 2.7.7.6 L O IT S O IN 2.7.1.52 te a n o l u g E S O CT A L A G 3.6.1.5 3.6.1.5 M IS L BO A T E M e s o ta n e p to l a m e n i te s y c 2.4.2.9 5.3.1.8 2.7.1.6 e s o c fu te a h p s o h -p -6 e s o n n a m te a h p s o h -p -6 e s to c u fr M IS L BO A T E M 2.3.1.7 1.3.8.1 1.3.99.12 1.3.99.- 1.3.8.7 2.3.1.13 2.3.1.13 1.3.8.7 1.3.99.12 1.3.8.1 2.3.1.7 1.3.8.7 1.3.8.4 2.3.1.13 2.3.1.7 1.3.1.2 e s to a g ta A o -C l y y r c a r d y h l y th -e 2 ET I D / T U G 2.7.1.33 l o r e c y l g l y to i m l a p i -d 2 , 1 te a r ta c a l a g 6.3.2.5 3.5.4.5 6.3.4.2 2.4.1.1 2.7.4.68 ) d i c a c i c u (m 3.1.3.66 2.7.1.154 2.7.1.154 3.1.3.36 3.1.1.25 3.1.1.17 3.1.3.56 e s to c a l a g 3.2.1.23 e s to c a l 4.1.1.21 2.7.1.48 2.7.4.14 2.7.4.6 2.7.1.7 2.7.1.1 3.1.2.4 te a r ta c a l a g e s o a tr te to l a m l to ti c a l a g e n i c y l g l y r ty u b l y th e -m 2 e n ti i n r a c l y r ty u b l y th e -m 2 CTP ) d i c a c i c u (m 2.7.1.1 ) l to i c l u (d e n i d ti y c MP -C ' 5 CDP 2.7.1.159 2.7.1.127 1.1.1.21 ) 5 (C e n to c a -l 4 , -1 o n o l u g mannose 1.1.1.48 2.7.7.6 te a l a x o te a l g ti A o -C l y l g ti 2.4.1.1 3.1.2.20 : C I T O I B O XEN : C I T O I B O XEN : C I T O I B O XEN : Y R A ET I D 3.1.3.5 3.6.1.5 3.6.1.5 - 5 , -4 l to i s o n i l y d ti a h p s o h p - 5 , 4 , -1 l to i s o n i - 5 , 4 , 3 , -1 l to i s o n i l to i s o n I 1.1.1.224 e n i te s y -c l y o n e th to n a p o h p s o h -p ' 4 A o -C l y l y r c a th e m A o -C l y n to o r c l y th e -m 3 e n ti i n r a c l y r e l a v o s i Normal metabolomics result) te a o i d e n a th (e te a n o e r th te a l y r c a r d y h l y th -e 2 e n ti i n r a c l y r ty u b o s i e n i c y l g l y r ty u b o s i 3.1.2.20 isovalerylglycine )P2 4 , (3 PI : C I T O I B O XEN : C I T O I B O XEN o c c a b To e n i th n Xa l a i r te c a b t u G t n a Pl te ta r a p s a 5'phosphoribosyl- te a h p s o h p i d te a h p s o h p i tr te a h p s o h p a tr te te a h p s o h p s i -tr 4 , 3 , 1 4.2.1.40 5.3.3.- 1 3.1.4.1 e s to c u fr e s o i tr to l a m Chemical Drug te i l o b ta Me m s i l o b ta Me conversion compoent te a l y x o b r a -c -4 e l zo a d i m i o n i m -a 5 4.2.1.42 1.1.1.17 1.1.3.8 2.4.2.4 2.4.2.1 - o n to c a l a g 1.1.1.31 te a n to o r c l y th e -m 3 1.17.4.1 2.7.7.8 e m o i b o r c Mi 4.1.1.36 te a h p s o h -p -1 l to i n n a m 2.4.1.1 3.1.3.86 3.1.3.56 3.1.3.62 e n to c a -l 4 , 1 te a n o c ta u l g l y th e -m 2 l i c a r u o r d y h i -d 6 , 5 2.7.1.151 ROS 3.1.2.20 2.7.1.74 y x o e -d -4 o r d y h e -d 5 e s to c a l l y l a i -s ' 6 glucose - to e -k ta e b c i n o l a m l y th -e 2 2.3.1.7 2.3.1.13 6.3.2.6 te a r a c u l -g -D e s to l a m semialdehyde te a n to c a l a g 4.2.1.17 6.4.1.4 2.3.1.13 4.2.1.17 3.1.3.67 1.1.1.45 2.7.1.2 ROS 3.1.3.22 te a n to c a l a g e n i e th te n a p o h p s o h p 4.2.1.17 e m o i b o r c Mi 3.1.1.25 e n i d ti y c y x o e d MP C d dCDP dCTP te a b r o c s a 2.4.1.1 2.7.4.14 2.7.4.6 , 4 , (3 PI e n i d ti y c y x o e -d ' 2 - 6 , 5 , 4 , -1 l to i s o n i ) C n i m ta i (V e n i d ti y c y x o e -d ' -2 l y th e -m 5 te a b r o c s a o r d y h e d l to i n n a m e n ti i n r a c l y l g ti 2.7.1.159 e n i c y l g l y o l g ti 3.1.3.74 te a h p s o h p o n o -m ' 3 te a h p s o h p s i k a tr te 6.4.1.3 3.5.2.2 5.5.1.4 3.1.3.9 3.1.3.25 4.1.2.20 1.2.1.3 3.1.3.5 3.6.1.19 1.8.5.1 2.7.7.3 A o -C l y r e l a v o s i y x o r d y -h 3 1.1.1.107 e n to c a l o x o d i r y -p 4 MP C -c ' 3 , ' 2 2.7.1.153 e n i c y l g l y n to o r c l y th e -m 3 5.3.3.- l to i s o n i - l y s o b i r o h p s o h -p ' 5 2.7.7.7 , e n o i th ta u l g , e n o i th ta u l g - -1 e s o c u l g te a n o i p o r p o d i e r u 4.1.1.34 te a h p s o h -p -6 e s o c u l g 3.1.3.74 3,4,5,6- A o C l y r ty u b - y x o r d y -h 3 - l y th e -m 2 )- e d i m a x o b r a c o n i c c u -(s 4 3.5.4.5 3.5.4.12 3.6.1.23 dUTP reduced d ze i d i x o te a h p s o h p 3.1.3.74 A o -C l y r ty u b o s i y x o r d y -h 3 A o -C l y n o c ta u l g l y th e -m 3 2.7.1.134 te a h p s o h p s i k a tr te te a r ty u b l y th e -m 2 A o -C l y r ty u b y x o r d y -h 3 n i m -a 5 2.7.1.151 ) SH (G ) SSG (G l y n o l a m l y th e 3.1.2.20 MP U -c ' 3 , ' 2 te a v u r y p 5.4.2.5 dephospho- te a r e l a v o s i y x o r d y -h 3 pyridoxine pyridoxine pyridoxamine - e l zo a d i m i o n i m -a 5 CoA 5.3.3.- 5.3.3.- - o n o c u l g o n o h p s o h -p 6 A e m zy n e o c ) 6 B n i m ta i (V e d i m a x o b r a -c 4 te a h p s o h p P PL te a h p s o h p pyridoxamine -P -1 l to i s o n i te a n o c u l g o h p s o h -p 6 1.4.3.5 1.4.3.5 pyridoxal 3.1.1.27 l to i s o n i l to i s o n i 3.1.1.31 e n to c a -l 5 , 1 1.1.1.49 5.3.1.9 2.3.1.7 2.3.1.13 1.1.1.21 3.5.1.6 2.7.4.6 pseudouridine l to i s o n i 1,3,4,5,6- a tr -te 6 , 4 , 3 , 1 deoxyuridine MP U d dUDP 3.1.2.20 4.3.2.2 2.7.1.21 2.7.4.9 DNA te a h p s o h p s i k te a h p s o h p s i k a x e h 2.7.1.158 te a h p s o h p s i k ta n e p 2.7.1.140 L A X O RID Y P l to i b r o s 3.1.2.4 1.1.1.178 1.1.1.35 3.1.2.20 4.2.1.18 5.3.3.- 2.3.1.7 2.7.1.35 2.7.1.35 te a h p s o h -p -6 e s to c u fr e m o i b o r c Mi 2.7.1.24 A BCA B6) t i (V te a x o d i r y p te a h p s o h -p 6 e n ti i n r a c l y r ty u b 1.2.3.1 e n i d ti y c l y th e -m 3 e n i c y l g l y r ty u b te a n o l a m l y th e IN V A L F RIBO 2.7.1.69 - l y s o b i r o h p s o h -p 5 1.1.1.44 1.1.1.10 M IS L BO A T E M M IS L BO A T E M 1.17.4.1 l to i b r o s - y x o r d y -h 3 - e l zo a d i m i o n i m -a 5 E IN ID RIM Y P 2.1.1.45 2 H D FA M IS L BO A T E M ) L r o (D l to i l y x e n i n a l -a ta e b OSE PENT xylulose l y r ta u l g l y th e -m 3 e d i m a x o b r a -c 4 2.4.2.7 1 2.7.1.1 1 3.1.3.1 M IS L BO A T E M e n i s to y c l y th e -m 5 te ta e c a to e c a l y th e -m 2 A o -C l ty e c a to e c a l y th e -m 2 CoA 2.7.7.7 TE PHOSPHA 3.1.2.20 te a n o c ta u l g l y th e -m 3 hydroxyisovaleroyl UDP 1.1.1.21 cadaverine te a r ty u b o s i y x o r d y -h 3 4.1.1.18 A o -C l y r ta u l g l y th e -m 3 e n ti i n r a c 3.6.1.9 3.1.3.2 AY W H T PA 2.7.1.1 l to i b i r 6 , -2 e s to c u fr 1.1.1.14 MP -A ' 3 MP A -c ' 3 , ' 2 te ta r a p s a e n i s to y c l y th e m y x o r d y -h 5 riboflavin te a h p s o h p s i -b te a h p s o h p s i -b 6 , -1 e s to c u fr e n i d i m y th MP T d flavin 3.1.3.46 2.1.2.3 3.1.3.5 3.6.1.5 dTDP 3.6.1.5 dTTP ribulose MP G c e d ti o e l c u n o n o m ) 2 B n i m ta i (V lysine 1.5.1.30 D FA te a h p s o h -p 5 ribulose 2.7.1.16 2.3.1.16 ) l y s o b i r o h p s o h -p -(5 -1 o d i m a m r -fo 5 5.3.3.- MP A c 3.1.4.17 te a h p s o h p o n o -m ' -3 e n i d i r u 1.1.1.35 1.1.1.31 e n ti i n r a c l y n o i p o r p 4.1.3.4 2.8.3.5 3.1.2.20 5.3.3.- b r a -c -4 e l zo a d i m i 1.1.1.21 te a r ty u b o s i o n i m -a 3 2.7.6.1 2.1.2.3 e n to e c a y x o r d y h i d 4.6.1.1 3.1.4.17 6.3.4.4 2.4.2.4 2.7.1.21 2.7.4.9 2.7.4.6 e s to c u fr te a n i c c u s o l y n e d a 4.1.2.13 L O RBIT O S 3.6.1.19 2.7.7.2 2.7.1.26 te a h p s o h p e n o n ta u -b 2 CoA succinyl e n i d ti y c l ty e c -a 4 N te a h p s o h -p -3 e d y h e d l a r e c y l g 4.6.1.2 P) A H (D 1.4.3.14 2.3.1.7 5.3.1.6 5.1.3.1 AND c i n o l a m l y th e m 5.3.3.- e s o b i -r D 2.7.1.15 acid 5.3.1.1 L RO CE Y L G 2.7.1.3 e n ti i n r a c l y r ta u l g l y th e -m 3 2.6.1.22 semialdehyde CoA propionyl te a r ta u l g l y th e -m -3 y x o r d y -h 3 e n i m y th o r d y h i d te a r ty u b o s i o d i e r u 1.2.1.27 RNA 1.3.1.2 3.5.2.2 3.5.1.6 te a o n ta u b o s i o n i m -a 3 e s o l u b i r o i th l y th e -m 5 - l y th e -m -5 to e k i -d 3 , 2 ) 6 (C e n i m y th ribose xylulose te a r ta u l g l y th e -m 3 4.3.2.2 c ti c a l to p a c r e -m 3 M IS L BO A T E M 1.5.1.8 2.7.4.8 te a h p s o h -p 1 te a h p s o h -p -1 l ty n e p o i th te a h p s o h -p 5 te a h p s o h -p 5 te ta e c a to e c a 4.2.1.109 3.1.3.77 2.2.1.1 2.7.1.17 te a o r p a c o n i m -a -6 to e -k 2 acid e s to c u fr 1.2.1.12 te a h p s o h -p 1 2.3.1.13 - e s o l tu p e h o d e s glyceraldehyde 1.2.3.1 1.2.1.3 2.7.1.29 e d ti p e p i D P AT ADP MP A MP I XMP MP G GDP GTP - to e -k -3 y x o r d y h i -d 2 , 1 te a h p s o h -p 7 te a h p s o h -p 3 2.7.7.6 3.6.3.14 2.7.4.3 3.5.4.6 1.1.1.205 6.3.5.2 3.6.1.6 2.7.4.6 2.7.7.6 5.4.2.7 6.4.1.3 1.53 1.13.1 e n te n e p o i th l y th e -m 5 - -4 s n a tr o n i m -a l ty e c -a N 5.3.1.23 N SPO e d ti p e p y l Po te a zo n e b y x o r d y -h 4 4.1.2.13 acids 1.14.13.33 1.14.99.15 1.5.5.2 hydroxyproline ribose saccharopine propionylglycine MP G -c ' 3 , ' 2 e n to e c a y x o r d y h i d RNA te fa l u s o i th 2.8.1.2 te a r e c y l g o h p s o h p i -b 3 , 1 te a h p s o h -p 1 te a n o l a m l y th e m c i tr e m y s s a e n i n i g r a l y th e m i d 1.1.1.27 1.17.4.1 1.17.4.1 2.2.1.2 A o -C l y n o l a m l y th e (S)-m e n i n i g r a l y th e m i d + MA (SD 5.3.3.- e s o b i r o i th l y th e -m 5 c i n o i p o r p o i th l y th e -m 3 2.2.1.1 1.54 1.13.1 te a zo n e b y x o th e -m 4 - e n i l o r r y -p 1 5.4.2.4 glyceraldehyde ) MA D (A e s a d ti o e l c u n e s a d ti o e l c u n c i v u r y p to p a c r e -m 3 te a h p s o h -p 1 1.14.13.12 2.7.1.31 ) MA D A 4.4.1.1 acid te a zo n e b y x o r d y h i -d 4 , 3 e s o r th y r e A o -C l ty e c a e n ti s y c - y x o r d y -h 3 l to i r th y r e 1.1.1.21 3.1.3.23 e n i e d i r e p i -p ta l e d e s a d ti o e l c u n 3.1.3.5 e n i te s y c o i th acid te a r e c y l g o h p s o h p i -d 3 , 2 te a l y x o b r a -c 5 2.7.2.3 te a l y x o b r a -c -2 5.1.99.1 dADP E T A O Z N BE 1.5.1.9 3.1.2.20 2.7.7.7 TP dA te a v u r y p 2.7.1.100 3.1.3.13 2.7.4.6 2.4.2.28 e s o r th y r e te a h p s o h -p -6 e s to c u fr dGDP dGTP y x o r d y -h 3 2.7.4.6 M IS L BO A T E M te a zo n e b te a h p s o h -p 4 te a zo n e b te a r u p p i h - -4 s n a tr 4.1.1.63 1.14.13.23 3.5.1.32 1.5.1.21 adenosine e n i s o th n a x guanosine 1.1.1.6 hydroxyproline 3.5.4.4 inosine 2.4.2.8 te a v u r y p l y n fi l u -s 3 c i o n ta u b o i th l y th e -m -4 o x -o 2 5.3.3.- te a r e c y l g o h p s o h -p 3 e n i l o r p y x o r d y -h -4 s i c N SPO 2.6.1.1 E RIN U P e n i n o i th e -m -L l y m r -fo N DNA acid te a r e l a v o n i m -a 5 2.7.4.8 DNA - o r th y r e 2.7.4.3 2.4.2.7 e d i x o i d r fu l u s te a m ta u l g y x o r d y -h 4 te a v u r y p y x o r d y -h o h p s o h -p 3 1.1.1.95 M IS L BO A T E M 2.6.1.1 2.7.7.7 l o h c te a c te a m ta u l g y x o r d y -h 4 ALDR ADR/ e n i s o n e d a o i th l y th e -m 5 semialdehyde 1.2.1.- ROS e s o b i r o i th l y th e -m 5 l o h c te a c n i te o Pr ) A (MT te fa l u s x to e D 1.14.99.23 A o -C l y n o l a m l y th e )-m (R e n i s o n e d a y x o e -d ' 2 1 5.4.2.1 - l y th e m i -tr 6 N te a p i d a o n i m -a 2 - e n i d i r e p i p o r d y h a tr -te 5 , 4 , 3 , 2 n o ti a d a r g e D e n i l o r p y x o r d y -h 3 2.4.2.1 2.4.2.1 2.4.2.1 MP G d lysine 3.1.3.5 3.5.1.31 2.6.1.5 semialdehyde te a l y x o b r a -c 2 e m o i b o r c Mi e n i te s y c 1 1.8.4.1 1.2.1.3 N SPO 1.5.3.7 te a l o c e p i p MP A d 2.6.1.52 3.1.3.5 2.4.2.1 glycerol e n i r u ta o p y h acid sulfinic ) 9 B t i (V te a l fo e n i n o i th e m e n i r e s o h p s o h -p 3 2.7.1.31 te a r ta u l g e n i r u ta 1.8.1.3 4.1.1.29 1.20 1.13.1 4.1.1.46 te a zo n e b y x o r d y h i -d 3 , 2 2.4.2.28 e d i x fo l u s te a r e c y l g o h p s o h -p 2 semialdehyde e n i s o n a u g y x o e -d ' 2 - y x o r d y -h 4 1.5.1.3 e n i r e s o h p s o h -p 3 e n i s y l l ty e c -a 6 N 2.6.1.23 te a r ta u l g o x -o 2 3.1.3.3 3.5.2.9 e n i n o i th e m 4.1.3.16 3.5.4.4 1.17.1.4 e m o i b o r c Mi alanine te a r ty u b o n i m -a 2 te a m ta u l g ROS E IN S Y L te a l fo o r d y h a tr te 1 4.2.1.1 5.3.3.- e n i te s y c te a l fo o r d y h i -d 8 , 7 te a l fo o r d y h a tr te e n e l y th e -m 0 1 , 5 2.7.1.165 IN E T RO P e n i th n a x o p y h e n i th n a x te a r e c y l g 3.5.4.3 guanine 2.4.2.1 M IS L BO A T CA - l y th e -m 1 - l y th e -m 3 d i c a c i to n a l l a E CIN Y L G serine te a r ta u l g te a v u r y p l o n e o h p s o h p N IO T A D RA G E D e n i d ti s i h e n i d ti s i h 1.17.3.2 E RIN E S ) te a o i d e n ta n e (p 2.4.2.1 3.4.13.18 (PEP) e n i s o n i y x o e -d ' 2 adenine n i to n a l l a e n i l o r p o x -o 5 phospho- 1.5.1.3 2.5.1.6 6.3.2.2 6.3.2.2 6.3.2.2 E IN N O RE H T 2.1.2.1 te a l o c y l g e n i s y l y x o r d y -h 5 e n i n o e r th IS S Y L CO Y L G e m o i b o r c Mi c i e n e g o n i te o Pr 3.5.3.4 ) te ta e c a y x o r d y (h te a l o c y l g o d i e r u M IS L BO A T E M 1.1.1.79 Acid Amino urea glycine e n ti i n r a c l y r ta u l g te a p i d a o n i m -a 2 te a p i d a o x -o 2 A o C l y r ta u l g 1.1.3.15 2.7.9.2 1.2.1.31 2.6.1.39 KGD 5.3.3.- ) 5 (C te a l fo o r d y h a tr te te a l y x o y l g e n i c y l g l y n i te s y c 2.6.1.2 - l y m ta u l -g a m m a g ROS sarcosine 2.1.1.20 1.1.1.27 - n i te o Pr - n i te o Pr 1.17.3.2 1.17.1.4 - o x -o -2 y x o r d y -h 5 e n i c y l g l y th e m i d glycine - l y m ta u l -g a m m a g e n i te s y c l y m ta u l -g a m m a G 1.5.8.4 ) e n i c y l g l y th e -m (N - l y m ta u l -g a m m a g e n i n o i th e m l y s o n e d S-a - l y th e m i -tr -N 6 - l y th e m i -d -N 6 - n i te o Pr H -1 o r d y h i -d 5 , -2 o d i e r -u 4 te a r ty u b o n i m -a 2 Acid Amino 1.5.8.3 te a v u r y p alanine alanine M) (SA te a l a x o lysine lysine e n i s y -l l y th e -m -N 6 te a r u o s i y x o r d y -h 5 e l zo a d i m i e n o i th ta u l -g e n i te s y c 2.6.1.45 4.3.1.17 te a n o c ta u l g - -. . 4 . 3 2.1.1.43 2.1.1.43 2.1.1.43 1.7.3.3 3.5.2.17 4.1.1.97 te a l fo o r d y h a tr te 5.3.3.- disulfide ) te a o i d e n a th (e 2.4.2.1 2.6.1.44 e n i s y -L n i te o Pr
6.3.2.3 6.3.2.3 6.3.2.3 2.1.1.13 2.1.1.- 2.1.1.5 A e m zy n e o c
c ti e c a e l zo a d i m i l y th e m 2.3.2.2 2.3.2.4 carnosine F H T -Me 5 acid 1.1.1.27 E IN IT RN CA te a r u 2.3.1.29 te ta c a l te a v u r y p y x o r d y -h ta e b l y r ty u b y x o r d y -h 3 1.1.1.81 High , e n o i th ta u l g IS S E H T N Y S BIO 1.1.1.35 CoA A o C l y o n to o r c A o -C l y n o c ta u l g e n i ta e b 4.2.1.17 1.3.8.6 1.3.99.7 reduced e l zo a d i m i l y th e m acid Amino e n i te s y c o m o h l y s o n e d S-a A o -C l ty e c a to e c a - y x o r d y -h 3 - o n i m a l y th e m i -tr -N 4 e d y h e d l ta e c a e n i m ta s i h l y th e -m 1 te a m l a th h p o r o n te a m l a th h p o e n o i th ta u l g l y th e S-m ) SH (G 1.2.1.5 1.4.3.4 2.1.1.8 2.5.1.18 ) H (SA 1.8 1.14.1 e n i s y l l y th e m i -tr -N 6 4.1.2.- e d y h e d l a r ty u b 1.2.1.47 l a x o y l g l y th e m C PD 1.1.1.103 te a o n ta u b o x -o -3 o n i m -a 2 E IN N IO H T E M 2.3.1.9 1 6.3.2.1 3.4.13.20 3.4.13.18 1.2.1.8 monamine - y x o r d y -h (S)-3 4.4.1.5 E IN E T S CY 4.1.1.32 e n i m ta s i h e n i n o e r th te ta e c a to e c a oxidase / oxidase 4.1.3.4 A o -C l y r ta u l g l y th e -m 3 2.3.3.10 E N IO H T A T U L G M IS L BO A T E M amine primary 4.1.1.28 3.3.1.1 1.8.1.7 e n i ta e b l a x o y l g l y th e m e n to e c a o n i m a e m o i b o r c Mi e n ti i n r a c o r d y h e -d 3 e n ti i n r a c y x o e d 1.1.9 1.1 ROS oxidase N SPO 6.4.1.1 - e l zo a d i m I e n i n a l -a ta e b e n i n a l -a ta e b e n i n a l -a ta e b M IS L BO A T E M e n ti i n r a c l ty e c a e n ti s y c o m o h aldehyde e d y h e d l ta e c -a 4 1.2.1.3 1.4.3.22 e n o i th ta u l g l y to c a S-l 4.1.1.4 c ti e c a e l zo a d i m I 2.3.1.7 4.1.1.22 , e n o i th ta u l g 1.1.1.30 acid 1.1.99.1 te a tr i c l y th e -m 2 e n i d ti s i h d ze i d i x o e n i te s y c o m o h E IN ID T IS H te a n a c o r -u s i c 1.8.4.1 e n ti i n r a c ) SSG (G 3.1.2.6 1.1.1.21 1.14.13.n7 1.1 1.14.1 M IS L BO A T E M te a r ty u b y x o r d y -h 3 A o C l ty e c a e m o i b o r c Mi 4.3.1.3 ) A B H (B 2.1.1.- te ta c a l choline E ET -H 2 1 ONE KET UV e n to e c a - -3 y x o r d y -h 3 E ET -H 1 1 2.6.1.58 e l zo a d i m i e m o i b o r c Mi 4.2.1.22 te a n ti o c i n y x o r d y -h 6 1 6.3.2.1 l to e c a S IE D BO te a r ta u l g l y th e m te a v u r y p l o i d e n a p o r -p 2 , 1 1.1.1.21 1.14.13.n7 3.1.2.27 NH3 1 6.3.2.1 e l zo a d i m i te a n ti o c i n E ET -H 8 -5 n i to n a d y h - l y m a b r a -c N l ty e c a to e c a anserine te ta c a l 2.4.2.1 ribonucleoside e n i d ti s i h l y th e -m 1 1.1.1.27 2.3.1.6 3.1.1.7 4.3.1.19 E T A IN T ICO N te a n a c o r -u s n a tr te a n o i p o r -p te a m ta u l g CoA 1.14.13.- ? e m o i b o r c Mi - y x o r d y -h 3 - o n i m -a 2 homocarnosine te a tr i c 2.3.1.9 AND 3.4.13.20 l y r ta u l g l y th e -m 3 e n i n o i th ta s y c te a n o c u m 4.2.1.3 1.5.1.- to e -k 6 5.3.3.- 2.3.3.1 2.3.3.10 CoA 1.1.12 1.1 1.1.9 1.1 e n i l o h c l ty e c a E ID M A IN T ICO N n i d n a l g ta s o r p te a r ty u b y x o r d y -h 2 E1 ) B H (A M IS L BO A T E M 1.1.9 /// ? /// 1.1.9 1.1 ? /// 1.1.9 1.1 - o n i m i m r fo 1.1.1.27 2.3.1.13 te a n ti o c i n 6.3.4.21 Low e l zo a d i m i 2.7.1.173 3.1.3.5 2.3.3.8 E PET -H 2 1 te a m ta u l g 4.2.1.49 te a n o i p o r p 3.5.2.7 2.1.2.5 te ta i n o c -a s i c 1.2.1.32 4.4.1.1 te a r ty u b o n i m -a 2 e n i m ta u l g E PET -H 8 E PET -H 1 1 A YP4 C / F YP4 C n i d n a l g ta s o r -p H -O 0 2 e n e i tr o k u e l - y x o r d y -h 0 2 te a tr i c o s i te ta e c a o l a x o H MD te a n o l a v e m 1.1.1.34 3.6.1.9 5.3.3.- 2 H n i d n a l g ta s o r -p H -O 0 2 G2 E ET o x -o 5 ) 4 (, d ) 4 (, -b e n e i tr o k u e l 2.3.2.- te a r ty u b to e -k a h p l a te a r u n ti o c i n 1.31 1.13.1 2.6.1.42 6.4.1.2 te a n i c c u s o n i n i g r a 4.1.1.6 te a n ti o c i n 1.33 1.13.1 2.7.1.36 te a n o c u m o n i m -A 2 to e -k 6 l y m ta u l -g a m m a g te a n o c u m y x o b r a c o n i m a 1.14.99.1 TE ALONA MEV d i c a c i n ti o c i n adenine te a n o l a v e m te a l i n a r th n a y x o r d y -h 3 semialdehyde n i d n a l g ta s o r p Acid Amino 1.6 1.13.1 semialdehyde 4.1.1.45 1.14.14.1 te a n o c ta i A o -C l y n o l a m e s o b i -r -D a h p l -a o h p s o h -p 5 e d ti o e l c u n o n o m e d ti o e l c u n i d F1alpha 1.1.1.- te a r a m fu M IS L BO A T E M te a h p s o h -p 5 4.3.2.1 6.3.4.5 te a h p s o h p i -d 1 ) MN a (N +) D A A (N 1.40 1.13.1 IDH ) te a n i c c u s e n e l y th e (m 2.4.2.19 2.7.7.18 6.3.5.1
1.33 1.13.1 1.14.99.1 1.14.99.1 2.3.2.2 1.14.13.30 asparagine 1.1.1.37 6.3.5.4 2.3.1.85 2.7.4.2 te a n e r u th n a x Amino acid Amino E ET -H 5 e n i n i g r a l ty e c -a N 5.3.3.- 3.7.1.3 te a m ta u l g e n a x o b m o r th 1.4.3.2 te a n o l a v e -m R glycine 1.14.13.39 OPHAN YPT TR A2 e n e i tr o k u e l te a h p s o h p i -d 5 e d i m a n ti o c i n o s i E ET -H 0 2 3.6.1.22 4 C e n e i tr o k u e l 4 B e n e i tr o k u e l N SPO e d i m a n ti o c i n te fa l u s E ET -H 9 ) 4 (, e te a r ta u l g y x o r d y -h 2 M IS L BO A T E M n i d n a l g ta s o r p 1.8.1.4 2.1.4.1 te a n i l o n i u q e d i m a n ti o c i n adenine I2 arginine e n i l l u tr i c alanine te ta e c a e l o d n i 2.6.1.1 IDH* 4.1.1.33 e d ti o e l c u n o b i r e d ti o e l c u n i d e d i x o c i tr i n +) D A (N e n i n e r u n y k y x o r d y -h 3 ) MN (N 2.7.7.1 1.1.9 1.1 l y n te n e p o s i 2.6.1.7 SO I 2.4.2.12 F L SU 4.4.1.20 3.3.2.6 e n i th i n r o E IN IN RG A te a l a m 5.3.99.5 te a r ta u l g to e -k a h p l a 2.3.2.- 1.14.13.34 te a h p s o h p o r y p A YP4 C / F YP4 C M IS L BO A T E M PP) (I 5.3.3.2 E ET -H 9 CA T te a n e r u n y k te ta e c a o n i d i n a u g 2.3.3.14 n i d n a l g ta s o r p 4.1.1.19 AND p c -a l y c a 5.4.99.2 te a tr i c o m o h E CL CY l y l l a l y th e m i d 5.3.99.4 H2 NH3 te a l i n a r th n a 1.2.1.3 E PET -H 5 4 A e n e i tr o k u e l CO2 1.4.1.3 te a h p s o h p o r y p 1.34 1.13.1 A RE U CoA propionyl te ta r a p s a 2.5.1.10 1.14.13.9 NADH PP) MA (D 3.5.3.1 2.1.3.3 2.4.2.1 e d i m a n ti o c i n E CL CY te a n o i p o r p e l o d n i l y x o d n -i 3 e d i m a n ti o c i n 3.1.3.5 3.7.1.3 te ta c a l e l o d n i e n ti a m g a riboside 1.14.14.1 2.1.1.2 geranyl te fa l u s e n e i tr o k u e -l y x o r d y -h 0 2 4.2.1.2 l y s e n r -fa s n a tr , s n a tr )- l y n e h p o n i m -a -(2 4 6.3.1.2 H D G -K a h p l a te a h p s o h p i d 4 F e n e i tr o k u e l E4 te a m ta u l g kynurenine e n i th i n r o te a h p s o h p i d te a o n ta u b o x o i -d 4 , 2 e d y h e d l ta e c -a -3 e l o d n I 2.7.1.22 -1.5 - +1.5 1.14.99.1 urea e n i m ta u l g 2.5.1.1 N SPO 2.6.1.7 1.6.1.2 lipoxin A4 lipoxin 6.3.4.16 S ID O N A S ICO E 3.3.2.- 3.5.1.2 2 G PG te a h p s o h -p l y o m a b r a c 1.1.1.188 5.3.99.2 e n ti a e r c 2.1.1.1 2.7.1.23 1 3.5.3.1 e m o i b o r c Mi F L SU 1.34 1.13.1 e n e a tr te y x o p -e 6 , 5 3.2.2.6 5.3.99.3 1.14.99.1 1.1.1.184 4.1.1.17 1.4.3.4 PH D A N 2.3.1.17 3.5.1.9 e s o b i P-r D A te a n o d i h c a r a 6.4.1.3 n i d n a l g ta s o r p n i d n a l g ta s o r p B4 lipoxin 2.5.1.21 1.33 1.13.1 E PET -H 5 1 3.3.2.- 3.5.1.15 n i d n a l g ta s o r p ) 6 n :4 0 (2 E IN T A CRE E IN M A Y L O P 4.1.1.15 CoA succinyl te a r a m fu 1.1.1.188 F2alpha 1.1.1.189 E2 2.7.3.2 squalene e n ti i n r a c l y n i c c u s e n e l a u q s y x o p -e 3 , (S)-2 D2 te a l e d n a m l y l l i n a v 1.14.13.132 2.6.1.13 te a v u r y p e l o d n i indole M IS L BO A T E M M IS L BO A T E M e d i m a n ti o c i n l y th e -m 1 2.3.1.7 6.2.1.5 ) (VMA 2.4.99.20 NAADP P+ D A N e n i n e r u n y k l y m r -fo N e n i m ta p y tr 5.3.3.- 1.1.1.141 e n i c s e tr u p 1 2.7.2.1 1.1.1.188 1.1.1.196 to e -k 5 1 2.5.1.16 te a r ty u b o n i m -a a m m a g n i d n a l g ta s o r p n i d n a l g ta s o r p e n ti a e r c A o -C l y n o l a m l y th e (S)-m 1.14.14.1 1.14.14.1 1.14.14.1 1.14.14.1 1.14.14.1 1.1.9 1.1 1.14.14.1 1.14.14.1 ) A B A (G 6.2.1.4 5.4.99.7 E2 1.3.5.1 J2 N SPO te a h p s o h p e n i n ti a e r c te ta r a p s a l ty e c -a N 1.2.1.5 te a n i c c u s 2.3.1.57 to e -k 5 1 n i d n a l g ta s o r -p ta e b 1 1 1.1.1.189 ) A A (N -P -5 te a m ta u l g 1.2.3.1 1.2.3.1 F2alpha n i d n a l g ta s o r p - l y th e -m 1 N 5.3.3.- 2.3.1.9 e n to e c a - l y th e -m 1 N l o r te s o n a l e m o i b o r c Mi F2alpha 2.6.1.29 1.3.1.72 1.4.3.2 - e n o d i r y -p 4 -EET 6 , 5 -EET 9 , 8 -EET 2 1 , 1 1 -EET 5 1 , 4 1 E ET )-H (R 6 1 E ET -H 5 1 A -EET 2 1 , 1 -1 H 5 1 A -EET 5 1 , 4 -1 H 1 1 e n i m r e p s l ty e c )-a (1 N 2.3.1.57 spermidine BA A G 1 1.1 1.13.1 4.1.1.28 - e n o d i r y -p 2 - -4 y x o th e -m 3 1.52 1.13.1 e d i m a x o b r a -c 3 e d i m a x o b r a -c 5 e m o i b o r c Mi e n i c s e tr u p l ty e c -a N l a n ta u b o d i m ta e c -a 4 5.3.3.- n i d n a l g ta s o r -p 2 1 ta l e d 1.4.3.4 te a m ta u l -g L 3.4.17.21 NAAGS te a n i c c u s SHUNT 1.1.1.1 hydroxyphenylglycolaldehyde 1.2.1.41 1.2.1.24 te a n i c c u s l y th e m i -d 2 , 2 J2 e d y h e d l a i m e -s a m m a g semialdehyde d o o (F 1.3.1.48 - l ty r a p s -a l ty e c -a N 1.2.1.88 ) e v ti i d d A l o r te s o n a l o r d y h i -d 5 2 , 4 2 1.3.1.48 3.3.2.10 3.3.2.10 3.3.2.10 3.3.2.10 1.1.1.232 1.14.14.1 1.14.14.1 2.5.1.22 1.2.1.3 4.1.1.4 n i to n a d y h l y th e -m N 1.5.3.16 te a m ta u l g 1.14.13.70 5.3.3.- - -4 y x o th e -m 3 te ta e c a e l o d n i y x o r d y -h 5 5.1.99.1 ) G A A (N te a o n ta u b o d i m ta e c -a 4 A o -C l ty e c a to e c a n a h p to p y tr e n i m ta p y tr y x o th e -m 5 e d y h e d l ta e c a e l o d n i y x o r d y -h 5 - 5 -1 o r d y h i -d 4 1 , 3 1 TE A T AR ASP 1 3.1.2.1 te ta e c a to e c a hydroxyphenylglycol 1.2.3.1 - l ty e c -a a m m a -g N - 5 1 , 2 1 , 11 1.4.3.4 ET H -D 5 , 5 ET H -D 9 , 8 ET H -D 2 1 , 1 1 ET H -D 5 1 , 4 1 5.3.3.- y x o e -d 5 1 n i d n a l g ta s o r -p to e k E ET -K 5 1 A ET H T - l y m r -fo -2 N to e -k 5 -1 o r d y h i -d 4 1 , 3 1 A ET H -T 5 1 , 4 1 , 1 1 spermine TE AMA GLUT n i d n a l g ta s o r -p 4 1 , 2 -1 ta l e d E2 3.5.1.63 SPO e n i m a n e r u n y k y x o th e -m 5 n i d n a l g ta s o r -p - ta s e l o h c l y th e m i -d 4 , 4 1.14.14.1 J2 M IS L BO A T E M te ta i m l a p 1.4.3.4 l o n e i -tr 4 2 , 4 1 , 8 glycine Molecule in library F2alpha ) 9 n :1 8 (1 te a e l o te a r ty u b o n i m -a a m m a g l to a k s n i n to o r e s ) :0 6 (1 3.7.1.2 2.1.1.6 ) A B A (G e s a r fe s n a tr l y th e -m O 1.5.1.2 E IN N A L A L Y N E H P e n i r h p e n ta e m 1.4.3.4 1.1.1.189 3.5.1.99 3.5.1.99 e n i m ta u l g A o -C l y n o l a m l y th e )-m (R 5.4.99.2 n i n to a l e m s d i p i l y x O 8 -1 C - e n i l o r r y -p (S)-1 E IN S RO Y T 1.3.1.70 3 n :5 0 2 e n i m ta u l g l ty e c a l y n e h p 5.3.3.- te a l y x o b r a -c 5 5.3.3.- e n i l o r p l ty e c -a N 2.3.1.14 te ta e c a to e c -a l y r a m -fu 4 l o c y l g e n e l y th e n e h p y x o r d y h i -d 4 , 3 1.14.16.4 n i d n a l g ta s o r p o r d y h i -d 4 1 , 3 1 5.3.3.- E IN L RO P proline M IS L BO A T E M 6 n :3 0 2 d i o n i b a n n a c o d En 2.3.1.37 F2alpha e n i m a p o d l y o n o d i h c a r -a N e n i m a p o d l y to i m l a -p N 3 n :6 2 2 te ta e c a l y n e h p y x o r d y -h 3 e n i m a p o d l y o e l -o N M IS L BO A T E M 1.5.99.8 n i n to a l e m y x o r d y -h 6 l y to i m l a -p N A o -C l ty e c a l y n e h p te a l l i n a v o m o h 1.52 1.13.1 e m o i b o r c Mi e n i r h p e n ta e m r o n 2.1.1.6 - a -5 l y th e m i -d 4 , 4 te fa l u s e l to a k s n i n to o r e s ID O BIN A N N CA O D N E 5.2.1.2 1.1.1.1 ) A V (H n i a h -c m u i d Me e n i r u ta n a h p to p y -tr -L y x o r d y -h 5 ) T H (5 n i n to o r e s l ty e c -a N te a e l o n i l 1.3.1.72 l -o -b -3 n e i -d 4 2 , -8 ta s e l o h c 4.1.1.28 2.3.1.87 2.1.1.4 2.3.1.9 2.3.1.85 y x o r d y -h 3 ) 6 n :2 8 (1 3.1.4.4 IS S E H T N Y S e n i r u ta 3.5.1.99 te a n i l u v e l o n i m -a 5 d i c a tty fa l y o r a te -s N 2.3.1.192 e n i c y l g l ty e c a l y n e h p te ta e c a to e c -a l y e l a -m 4 - y x o r d y -h 3 te ta s i r y m n i a h c t r o h s e n i r u ta te ta i m l a p y x o r d y h i -d 4 , 3 e m o i b o r c Mi ) :0 4 (1 te a o n a p o r p l y n e h -p 2 te ta e c a l y n e h p 1.14.13.72 2 d s n a tr c s y x o r d y -h 3 l ty e c a to e c a ) :0 8 (1 te a r a te s ) :0 6 (1 mandelaldehyde epinephrine te ta c a )l l y n e h p y x o r d y -h -(4 3 4.2.1.24 but not detected - a -5 l y th e m i -d 4 , 4 A o -C l y o n e A o -c l y c a 1.3.8.1 4.2.1.17 1.1.1.35 CoA e m o i b o r c Mi te ta c a l l y n e h p l -o b -3 n -e -8 ta s e l o h c fibrinogen 1.5 1.13.1 1.2.1.5 I Uroporphyrin ) A (PL te ta e c a l y n e h p y x o r d y -h 2 - l y th e -m b -4 y x o b r a -c a 4 3.1.2.2 o h p s o h p i -d ' -5 e n i d ti y c 2.1.1.6 fibrinogen l o r e c y l g l y c a i Tr te ta e c a l y n e h p y x o r d y -h 4 2.1.1.6 e n i m a l o n a th e l -o b -3 n e i -d 4 2 , -8 ta s e l o h -c a 5 L RO E T S E L O CH B e d ti p e p e n i m a l o n a th e e n i m a l o n a th e o h p s o h p 6.2.1.3 1.2.1.5 3.4.21.5 EG D porphobilinogen ) G A (T 2.7.8.1 2.7.7.14 2.7.1.82 6.2.1.3 3.1.2.2 3.1.1.4 6.2.1.3 AND 1.4.3.4 2.1.1.28 medium chain medium 3.1.1.3 - y x o th e -m 3 e s a d ti p Pe fibrinogen te a s ti n e g o m o h K SF XVPG SPVPD ROS n i a h t-c r o Sh e m o i b o r c Mi -D IN M A IT V y x o r d y -h 3 l y n e h p y x o r d y -h 4 A e d ti p e p N E G O IBRIN F R XSXA VD K SD D T T A 2.5.1.61 A o -C l y c a e n i d ti y c choline te a d i h c a r a A o -C l y o -En 2 -D s n a tr A o -C l y c a A o -C l y c a to e -k 3 1.14.19.3 e m o i b o r c Mi 1.27 1.13.1 e d y h e d l ta e c a 4.2.1.17 1.1.1.35 2.3.1.16 e n i l o h c o h p s o h p i -d ' 5 choline e d y h e d l ta e c a l y n e h -p 2 1.1.1.170 IS S E H T N Y S 2.7.8.2 2.7.7.15 te a h p s o h p 2.7.1.32 ) :0 0 (2 A o -C l y o r a te s A o -C l y to i m l a p E G A V A E CL E3 / E1 E3 / E1 te fa l u s l o n e h p 1.2.1.5 te ta e c a l y n e h p y x o r d y h i -d 4 , 3 A o -C l y to s i r y m e s a d ti p Pe e s a d ti p Pe S E ID T P E P e s a d ti p Pe e s a d ti p Pe 2.3.1.20 uroporphyrinogen 2.3.1.13 9 n :2 8 1 1.27 1.13.1 noradrenaline e s a d ti p Pe l o tr te i c l a c l o i tr i c l a c I e n a l i b l y th e m y x o r d y h te a v u r y p l y n e h p 1.14.13.126 1.14.13.13 4.1.1.37 N SPO l o r te s o m zy l y th e -m -4 to e -k 3 e s a d ti p Pe 2.3.1.137 F L SU VR G G SEG L EF G K ED T D T 1.4.3.4 R XSXA VD K SD D T T XSXA VD K SD D T T A 1.3.8.7 1.14.19.1 1.2.1.3 ) 9 n :1 8 (1 te a e l o 1.14.19.1 1.14.19.1 calcidiol XR D G G XEA SEF T T G T D A e s a d ti p Pe 4.2.1.75 te a v u r y p l y n e h p y x o r d y -h 4 -) l y n e h p y x o r d y -h (4 n i a h t-c r o Sh Chain Long e d y h e d l ta e c a VR G G EG XA F D EG SG D A l to i s o n i l y d ti a h p s o Ph e n i l o h c l y d ti a h p s o Ph Diacylglycerol 1.4.3.4 l o r fe i c l a c e s n i a h t-c r o Sh acylglycine e n i m a l o n a th e l y d ti a h p s o Ph 1.14.13.126 VR G G EG XA F D EG SG p D A coproporphyrinogen d i c A tty a F phenol s d i p i l o s Ly ) (PI ) (PC ) G A (D E5 1.1.1.270 e s a d ti p Pe e s a d ti p Pe e s a d ti p Pe e n ti i n r a c l y c a (PE) Chain Long E3 / E1 y x o r d y h i -d 4 , 3 e s a d ti p Pe e s a d ti p Pe I e n i m a l y th e l y n e h -p 2 uroporphyrin s e s a r fe s n a tr l y th e -m PE-N / A o -C l y c A 3.1.2.2 A o -C l y o e l o e d y h e d l ta e c a l y n e h p 1.14.13.159 n i a h -c m u i d Me A o -C l y o e l to i m l a p a o -c l y o e l to s i r y m III Light I I -I n e g o n i r y h p r o p o r u Long Chain Long te a n e h e b 1.4.3.4 2.6.1.5 A o -C l y c a 2.6.1.1 1.4.3.2 1.4.3.2 l o r te s o m zy l -o b -3 n -e t-8 s e l o h -c a 5 V G G SEG L EF G K ED T D T 2.3.1.137 9 n :3 0 2 1.14.19.- 9 n :2 0 2 1.14.17.1 1.3.1.72 e s a d ti p Pe A PL s d i c A tty a F serine ) :0 2 (2 3 D n i m ta i v R XSXA VD K SD D XSXA VD K SD D T T e m o i b o r c Mi XR D G G XEA SEF T T G T D 3.1.3.4 e n i m a r ty e s a d ti p Pe l o r te s o m zy l y th e -m a 4 3.1.2.2 3.1.2.2 4.1.1.37 n i a h C m u i d Me e n i m a r ty y x o th e -m 3 A e m zy n e o c 2.7.8.29 2.7.8.- 4.1.1.53 5.3.3.5 VR G G EG XA F D EG SG D 5.3.3.5 e s a d ti p Pe d i c A tty a F 5.3.3.- e s a d ti p Pe e m o i b o r c Mi E3 / E1 3.1.2.2 1.4.3.4 ROS 2.3.1.50 ID IP L O G IN H P S coproporphyrin coproporphyrinogen 2.3.1.16 E3 / E1 l o r te s o m s e d o r d y h e -d 7 6.2.1.2 te a e l to s i r y m dihydoxyphenylalanine 1.3.1.72 III III n i a h -c m u i d Me 5.3.3.- ROS e n i r e s l y d ti a h p s o Ph ) 5 n :1 4 (1 XSXA VD K SD M IS L BO A T E M phenylalanine 1.14.16.1 e n i s o r ty 1.14.16.2 ) A P O -D (L 4.1.1.28 dopamine 2.1.1.6 e n ti i n r a c l y c a sphingosyl o r d y h i -d o s y l Molecule not in (PS) E3 / E1 - ta s e l o h -c a 5 d ze i d i x O sphingomyelin te a n e c c a -v s i c l o r te s e l o h c e n i n a g n i h p s to e -k 3 phosphorylcholine te a o d n o g te a e l to i m l a p l o r te s o th a l l o r te s e l o h c o r d y h e -d 7 2.8.2.2 12/15 LOX 12/15 5.3.3.- l -o b -3 n e i -d 4 2 , 7 1.3.1.72 1.14.21.6 1.3.1.21 A o -C l y c a to e -k 3 6.2.1.3 s d i p i l o h p s o Ph te a r e c o n g i l ) 7 n :1 8 (1 te fa l u s ID IP L O H P S O H P S N R S O R 3.1.4.12 )) -9 (n :1 0 (2 ) 7 n :1 6 (1 1.3.3.3 te a d ti a h p s o -Ph L ) :0 4 (2 coproporphyrin 3.1.1.5 3.1.6.2 M IS L BO A T E M I s n i l e y m o g n i h Sp 2.7.8.27 1.1.1.102 3.5.1.109 1.3.1.72 l o r e c y l g l y c a i P-d D C glycerophosphorylcholine 1 2.7.8.1 2.7.7.41 e n i s o r -ty -L o d o i i -d 5 , 3 ID RO Y H T n e g o n i r y h p r o p to o r p 1 1.1.1.21 l y r te s e l o h C n i a h -c g n o L e n i m a l o n a th e o h p s o h p o r e c y l g ) PC (G e n to e c a y x o r d y h i d 3 n :3 8 1 6 n :2 8 1 2.3.1.43 l o r te s o m s e d IX l y s o g n i h p -s o r th y r e ) 4 (T e n i x o r y th E N O RM O H r te Es N IO T A ID X -O A T BE A o -C l y c a 2.3.1.51 te a n o v r e n te a c u r e te a h p s o h p 1.1.8 1.1 l o r te s e l o h c d i c a tty fa phosphorylcholine Ceramides 5.3.3.- ) 9 n :1 4 (2 ) 9 n :1 2 (2 P) A H (D - -. . 4 1 . 1 E3 / E1 E3 / E1 IS S E H T N Y S 2.3.1.26 sphinganine 1.3.3.4
1.14.19.3 e n i s o r -ty -L o d o i i -d 5 , 3 1.1.8 1.1 e n i s o r ty o d o -i 3 1.1.8 1.1 e s a l o r d y -h PC -G l y n e k l a n i a h -c g n o L 3.1.2.2 - -SN l y c A plasmalogen / y x o r d y -h 3 3.1.4.2 3.1.4.2 e n i s o g n i h p s to y h p e n i n o r y th o d o i i -tr ' 3 , 5 , 3 n i r y h p r o p to o r p 1.3.8.8 n i a h -c g n o L n i a h -c g n o L P -3 l o r e c y l g e s a th n y s d i c a tty fa - 2 S-D N A R T 2.3.1.24 3.5.1.23 ) 3 (T library A O -C YL C A XY O R YD -H -3 L 2.7.8.5 3 :n 8 1 6 n :3 8 1 1.1.8 1.1 IX 1.14.13.17 1.14.99.38 1.14.13.98 A O -C YL O EN A o -C l y c a e n ti i n r a c l y c a Y T T FA y x o r d y -h b 2 2 4.2.1.17 2.3.1.21 glycerol 6.2.1.3 2.3.1.21 3.5.1.23 e n i s o g n i h p s l y s to c a l a g e n i s o r ty o d o -i 3 2.4.1.23 l o r te s e l o h c 1.14.13.15 te a h p s o h -p 3 sphingosine CID A n e g o l a m s a Pl 1.3.8.9 P) 3 (G Cardiolipin 1.14.15.6 1.14.15.6 2.7.8.- s d i c A tty a F M IS L BO A T E M E5 4.99.1.1 - l y d ti a h p s o h -p -1 L dihydroceramide 2.3.1.15 -P l o r e c y l g 5.3.3.- l o i -d 6 2 , ta e b -3 e n -e t-5 s e l o h c l o r te s e l o h c y x o r d y -h a h p l -a 7 l o r te s e l o h c y x o r d y -h 5 2 l o r te s e l o h c y x o r d y (S)-h 4 2 y x o e d o r d y h a tr te - l y d ti a h p s o h -p -1 L 2.3.1.24 3 n :4 0 2 6 n :3 0 2 y x o r d y h i -d b 2 2 , a 0 2 e n o r te s o c ti r o c glycerol l y th e m i -d N , N 1.1.1.213 heme 3.1.3.27 l o r te s e l o h c - -. . 4 1 . 1 allopregnane- 2.7.1.30 sphingosine 3 n :6 4 2 3 n :5 4 2 6 n :4 2 2 3 n :5 0 2 3.1.3.4 2.7.1.91 3 n :6 2 2 e n o i -d 0 2 , -3 l -o 1 2 pregnanolone pregnanediol 1.1.1.53 1.1.1.50 Circle size relative to Z- E5 / E2 1.14.19.- a g e m o tty fa x -o ta e b 1.14.19.3 E5 / E2 hemoglobin/heme te a n o d i h c a r a hydroxy acid hydroxy 1.14.15.3 6 n :5 4 2 6 n :4 4 2 s n i te o r p glycerol 6 n :5 2 2 3 n :5 2 2 ) 6 n :4 0 (2 1.14.13.15 1.14.13.100 1.14.13.17 1.1.1.181 1.14.13.100 1.14.15.6 1.14.13.99 y x o r d y -h a h p l a 0 2 1.14.15.6 - ' -5 e n i d ti y c l o r te s e l o h c 2.7.7.39 - e n i s o g n i h p -s o r th y r e 1.3.1.30 diphosphoglycerol te a h p s o h -p 1 e n o l o n a l o h c o ti e BIN O L G O M E H e n o r te s o n e r d a - y x o r d y h i -tr 1 2 , b 1 1 , a 3 e n o i d e n a l o h c o ti e e n o l o n a l o h c o ti e glucuronide l -a 8 -1 n a n g e r -p b -5 o x -o 0 2 1.1.1.50 AND e n o tr s e y x o th e -m 2 e n o tr s e - e n -e t-5 s e l o h S)-c 4 (2 1.14.99.3 IC L Y X RBO ICA D te fa l u s e n o tr s e te a o n te s e l o h -c -5 y x o r d y -h ta e -b 3 e d i n o r u c u l -g 3 glucuronide 1.1.1.146 - 0 2 , -3 y x o r d y h i -d 1 2 , b 1 1 y x o r d y -h 8 1 l o r te s e l o h c y x o r d y h i -d 7 2 , a h p l -a 7 e n -o -3 e n te s e l o h -c y x o r d y -h a 7 l o r te s e l o h c y x o r d y h i -d 5 2 , -a 7 l o i -tr 4 2 , a h p l a 7 , ta e b 3 RIN Y H RP O P 1.1.1.1 allopregnanolone l -a 8 -1 n a n g e r -p b -5 o x o e n o r te s o d l a e n o r te s o c ti r o c 1.1.1.213 CID A 1.3.1.3 1.1.1.50 2.4.1.17 1.3.1.3 1.14.15.4 1.14.15.5 e n o i -d 0 2 , -3 e n a n g e r -p ta e b 5 M IS L BO A T E M - y x o r d y h i -d 1 2 , a 3 ID RO E T S LC M IS L BO A T E M e n o i -d 0 2 , 1 -1 e n a n g e r -p b 5 te a l y x o b r a c i d E N O RM O H score 2.4.1.17 A o -C l y c a biliverdin t- s o r d n a y x o r d y -h b 1 1 IS S E H T N Y S BIO 1.1.1.181 e n o i -d 0 2 , -3 e n a n g e r -p a 5 1.14.13.100 2.8.2.4 e n o i -d 7 1 , -3 e n -e 4 1.1.1.181 1.1.1.181 a g e m o tty fa 2.4.1.17 - b -5 y x o r d y -h 1 2 o r d y h e -d 1 1 e n o i -tr 0 2 , 1 1 , -3 e n a n g e r p aldo acid aldo 1.1.1.50 5.3.3.- e n o r te s o c ti r o c 1.3.1.3 - n a n g e r -p a h p l a 5 1.14.99.9 1.14.13.96 1.3.8.8 e n o tr s e y x o th e -m 2 e n -o 3 , l -o a h p l a 0 2 1.3.1.30 e n o r te s o r d n a 1.1.1.149 3 7 1.3.1.24 ROS glucuronide 1.14.15.6 - a h p l -a 7 , ta e b 6.2.1.3 e n o i -d 7 1 , -3 e n ta s o r d n -a a 5 e n o r te s o r d n a 1.1.1.146 - y x o r d y h i -d 6 2 , a h p l -a 7 - y x o r d y h i -d 5 2 , a h p l a 7 - y x o r d y h i -d 4 2 , a h p l a te a o n te s e l o h -c -5 y x o r d y h i d 1.14.15.4 1.3.1.3 e n -o -3 n te s e l o h -c 4 E BIL e n -o -3 n te s e l o h -c 4 e n -o -3 n te s e l o h -c 4 e n o tr s e y x o r d y -h 2 E) (E, n i b u r i l i b 1.14.14.1 1.2.1.3 2.1.1.6 1.3.1.22 1.1.1.50 2.4.1.17 CID A , a h p l a -2 n te s e l o h -c 4 M IS L BO A T E M ) Z , (Z n i b u r i l i b t- s o r d n a o x -o 9 1 e n -o -3 l o i -d a h p l a 2 1 1.1.1.- d i o r te s n g e r p e n o tr s e e n o i -d 7 1 , -3 e n -e 4 1.1.1.146 y x o r d y -h 1 2 te a n i c c u s 1.14.14.1 te a p i d a 1.14.15.4 e n o r te s o c ti r o c y x o e d te fa l u s o n o m Z (E, n i b u r i l i b I SD / SD -H -b 3 pregnenolone N SPO A o -c l y o -En 2 -d s n a tr 1.1.1.181 e n o r te s o c ti r o c o r d y h a tr te 1.14.99.10 E) , Z r o 1.1.1.50 1.14.13.15 1.1.1.50 1.14.13.15 1.14.13.15 1.14.13.15 6.2.1.7 1.3.1.3 1.1.1.50 1.14.13.15 1.14.13.15 1.14.13.15 y x o r d y h i -d a 0 2 , a 7 1 l a n ta n e p l y th e -m 4 2.4.1.17 l o r te s e l o h c t- s o r d n a y x o r d y -h a 6 1 n i a h -c m u i d Me e n o tr s e y x o r d y -h a 6 1 n i a h -c m u i d Me 1.14.14.1 1.14.14.1 e n o i -d 7 1 , -3 e n -e 4 y x o r d y -h 3 N SPO te a l y x o b r a c i d 3.1.6.2 2.8.2.2 te a l y x o b r a c i d 1.14.99.10 1.1.1.35 4.2.1.107 1.17.99.3 5.1.99.4 1.1.1.35 4.2.1.107 1.17.99.3 5.1.99.4 6.2.1.7 d i c a tty fa n i a h -c g n o L - -3 y x o r d y -h a h p l -a 7 d i c a tty fa 2.3.1.16 - y x o r d y h i -d 1 2 , a 7 1 te a l y x o b r a c i d e n o s ti r o c o r d y h a tr te te a o n te s e l o h -c -4 o x o bilirubin e n o i -tr 0 2 , 1 1 , -3 e n a n g e r -p b 5 1.1.1.50 2.3.1.176 e d i n o r u c u l g i -d ta e b d i c a tty fa t- s o r d n a y x o r d y -h 9 1 2.3.1.176 4.2.1.17 y x o r d y -h b 1 1 1.1.1.62 e n o i -d 7 1 , -3 e n -e 4 1.14.14.1 e n o i d e n te s o r d n a e n o r te s o c ti r o c e n o r te s e g o r p e n o r te s e g o r p pregnenolone A o -C l y o l o h c y x o e d o n e h c A o -C l y o l o h c l o s ti r o c o r d y h a tr te 1.1.1.50 1.14.99.10 1.14.15.4 I SD / SD -H -b 3 l o i tr s -e e d i n o r u c u l -g 6 1 1.1.1.62 A o -C l y c a to e -k 3 - y x o r d y h i -d 1 2 , b 1 1 e n o i -d 0 2 , -3 e n a n g e r -p b 5 I SD / SD -H -b 3 1.14.15.6 y x o r d y -h 3 C L t- s o r d n a y x o r d y -h 9 1 1.3.1.3 1.14.99.9 te a l y x o b r a c i d o r d y h e d y x o r d y -h a 6 1 3.2.1.31 e n o i -d 7 1 , -3 e n -e 4 o r d y h i -d a 0 2 A o -C l y c a e n o r te s o r d n a o s i 2.4.1.17 l o i tr s e - y x o r d y h i -tr 1 2 , a h p l a 7 1 , ta e b 1 1 1.14.99.9 6.2.1.7 3.1.2.27 2.3.1.65 2.3.1.65 2.3.1.65 2.3.1.65 3.1.2.27 6.2.1.7 1.14.14.1 l o r te s e l o h c y x o r d y -h a h p l -a 7 1.14.99.9 e n o r te s e g o r p 6.2.1.3 1.14.13.17 - 0 2 , -3 e n a n g e r -p ta e b 5 1.1.1.149 y x o r d y -h a h p l a 7 1.1.1.35 pregnenolone 1.14.13.100 3.1.2.2 1.14.14.1 1.14.14.1 y x o r d y -h 7 1 5.3.3.- e n o r te s to s te o x -o 9 1 l o i d a tr s e 1.14.14.1 l o s ti r o c y x o e -d 1 2 e n o r te s e g o r p e n o l o n e n g e r p y x o r d y -h a 7 1 1.14.15.4 I SD / SD -H -b 3 o n e h c o r u ta glycocheno urobilinogen 1.1.1.239 1.1.1.64 I SD / SD -H -b 3 te a l o h c y x o e d te a l o h c y x o e d te a l o h c o c y l g te a l o h c o r u ta
l o i d a tr s e y x o r d y -h 2 1 1.1.1.21 2.1.1.6 1.14.14.1 dehydro y x o r d y -h 3 MC 1.3.1.3 te a l o h c i r u -m ta e b te a l o h c i r u -m a h p l a x to e D te a n e l o h c o r u ta MC e n o r te s o r d n a i p e 5.3.3.- te a n e l o h c o c y l g 1.14.99.10 1.14.99.10 e m o i b o r c Mi te a l y x o b r a c i d x to e D te fa l u s te a l y x o b r a c i d te fa l u s A o -C l y c a 1.14.14.1 e m o i b o r c Mi A o -C l y c a ? l o i d a tr s e y x o th e -m 2 e m o i b o r c Mi e m o i b o r c Mi e m o i b o r c Mi e m o i b o r c Mi 1.14.15.4 e n o l o x te r o c I SD / SD -H -b 3 1.14.99.10 t- s o r d n a y x o r d y -h a 7 3.1.6.2 mesobilirubinogen A o -C l y c a to e -k 3 1.14.14.1 e n o r te s to s te y x o r d y -h a 7 1.14.14.1 e n o i -d 7 1 , -3 e n -e 4 1.1.1.146 - g e r p y x o r d y h i -d 1 2 , a 7 1 n i l i b o r -u D 1 1.14.99.1 2.8.2.15 nenolone te a l o h c o y h 1.14.13.97 2.4.1.17 e n o r te s to s te l o s ti r o c 4.2.1.17 e n o r te s to s te y x o r d y -h 9 1 1.14.14.1 e n o s ti r o c 2.4.1.17 e m o i b o r c Mi 1.1.1.- y x o r d y h i -tr 1 2 , a 7 1 , b 1 1 te a l o h c y x o e d o c y l g te a l o h c y x o e d o n e h c te a l o h c pregnenolone te a l o h c o th i l o r u ta 2.3.1.16 I SD / SD -H -b 3 1.14.15.4 n e g o n i l i b o c r te -s L b 7 -1 l o i d a tr s -e y x o th e -m 2 ta e b 7 -1 l o i d a tr s e l o i d a tr s -e ta e b y x o r d y -h ta e b 6 e n o r te s to s te o r d y h i d l o i -d ta e b 7 1 , a h p l a -3 n ta s o r d n -a ta e b 5 1.3.99.5 1.1.1.50 te fa l u -s 3 Y R A D N CO E S 1.3.8.7 e d i n o r u c u l -g 3 e d i n o r u c u l -g 3 te fa l u -s 3 ta e b 7 -1 l o i d a tr s e te a l o h c o th i l o r u ta e m o i b o r c Mi 5.3.3.- - 2 -D s n a tr e m o i b o r c Mi E BIL A o -C l y o En 4.1.2.30 S CID A e n o r te s to s te e m o i b o r c Mi e m o i b o r c Mi e m o i b o r c Mi ? te a l o h c y x o e d te a l o h c y x o e d o s r u x to e D te a l o h c o th i l e m o i b o r c Mi e m o i b o r c Mi e n o r te s to s te o r d y h i -d b 5 1.3.1.3 2.4.1.17 glucuronide te fa l u s EA H D 2.8.2.2
n i l i b o r -u L 4.1.2.30 te a l o h c o th i l o c y l g e m o i b o r c Mi te a l o h c o th i l o c y l g te fa l u s e m o i b o r c Mi
te a l o h c i r u -m a m m a g te a l o h c i r u -m ta e b te a l o h c o s r u te a l o h c y x o e d o r u ta c. n I , n o l o b a t Me 5 1 0 2 t h g yri p o C 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 # of samples •128 from patients with diagnosis of IEM fatty acid… •72 “Normal Controls” cofactor…
creatine •27 different IEMs other branch chain aa •Majority of patients organic acidemia on treatment Urea cycle unknown
0 10 20 30 40 50 60 70 80 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
•Intra-assay precision •median=10.47% (IQR= 5.55- 22.04%)
•Linear detection •34 acylcarnitines and amino r = 0.94 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 • Organic acidemias – Arginase def – 3-methylcrotonyl-CoA – Argininosuccinate lyase def carboxylase def – Citrullinemia – Cobalamin disorders – Ornithine transcarbamylase def – Glutaric acidemia type I • Amino acid disorders – HMG-CoA lyase def – Homocystinuria (CBS) – Isovaleric acidemia – Maple syrup urine disease – Methymalonic acidemia – Phenylketonuria – Propionic acidemia • Fatty acid oxidation disorders • Other – MCAD – Guanidinoacetate methyltransferase def – VLCAD – Holocarboxylase synthetase def – Thymidine phosphorylase def – TMLHE def results Neurologic phenotypes FUNCTIONAL VALIDATION OF DNA VARIANTS OF UNCERTAIN SIGNIFICANCE Case 1 – Citrate Transporter EXOME Findings Deficiency • trans mutations in SLC13A5 •c.997C>T (p.R333X) and c.680C>T (p.T227M) •Disorder: AR citrate transporter def.
Citrate
TCA cycle 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
VMA Z score Z
Patient is shown in red Dopa-containing Aromatic amino acid medications decarboxylase deficiency
High
Low
-1.5 - +1.5
Molecule in library but not detected
Molecule
Circlenot size in relative to Baylor Genetics Z-scorelibrary Case 3
• 19 month old male • Prior normal workup – Global developmental – Microarray delay – Metabolic workup – Hypotonia • Plasma amino acids – Abnormal movements • Lactate – Abnormal MRI (delayed • Ammonia myelination) • Urine organic acids – Oculomotor apraxia • CSF amino acids – Facial hemangioma • CSF neurotransmitter – Constipation 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
Imidazole propionic acid UROC1
Histidase Urocanase
Histidine Urocanic acid Imidazolone propionic acid
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] GABA transaminase (ABAT) deficiency (Plasma!)
2-oxoglutarate 2-pyrrolidinone 6 Glutamate 66
4 Glutamic acid decarboxylase 44
2 22 2-pyrrolidinone
GABA
Z score Z 0 00
GABA-transaminase
2 2 - -2-
Succinic semialdehyde
4 4 - -4-
Plasma PlasmaPlCa smaSF(n=124) CSF Succinate Patient samples
2-pyrrolidinone - a new biomarker for ABAT deficiency 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 to Genomics
Diagnosis Contribution of Metabolomics
37.8% 62.2% 50.5% 49.5%
Provided Information Did not aid in the case
Molecularly Solved Contribution of Metabolomics to Genomics
Contribution of Areas Improved Metabolomics 19.1%
37.8% 14.7% 62.2% 75%
Provided Information Rules Out Variants from Diagnosis Did not aid in the case Variant Classificaiton Change
Confirms Molecular Diagnosis Contribution of Metabolomics to Variant Interpretation
Variant Classification Changes Areas Improved B LB VUS LP P 19.1% 6 14.7% 5 75% 2
1 Rules Out Variant from Diagnosis 1 Variant Classification Change
Confirms Molecular DiagnosisB = benign LB = likely benign VUS = variant of uncertain significance LP = likely pathogenic P = pathogenic Peroxisomal Biogenesis disorders (PBD) FURTHER VALIDATION AND NEW DISCOVERIES PBD are a clinical spectrum of disease Disease Phenotype Classic Zellweger Neonatal Adrenoleukodystrophy Infantile Refsum
Disease Severity Biochemical Phenotype ↑ ↑ VLCFA’s ↑ or normal VLCFA’s Absent or deficient peroxisomes Reduced # peroxisomes PEX alleles Severe hypomorphic or null Mild hypomorphic
Pictures from Inborn Metabolic Diseases 4th edition and SIMD NAMA 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
Zellweger-spectrum disorders Metabolomics ~650 named molecules identified in each plasma sample, N=19
Relative increase 20
Amino Acids Relative decrease 10
Peptides 0
Carbohydrates
-10 Energy Long-chain Dicarboxylic Acids
-20 Phosphatidylcholines (PC) Lipids
Phosphatidylethanolamines (PE) -30 Component Component [10.10%] 2
Plasmalogen phospholipids -40 Sphingolipids
-50 -40 -30 -20 -10 0 10 20 Nucleotides Cofactors Component 1 [18.98%]
Xenobiotics PBD Controls Results: Untargeted metabolomic analysis Pipecolic Acid C24 (VLCFA) 7-HOCA (bile acid) Hexadecanedioate Octadecanedioate Eicosanodioate Docosadioate 1-(1-enyl-palmitoyl)-GPC* 1-(1-enyl-oleoyl)-GPC* 1-(1-enyl-stearoyl)-GPC* 1-O-hexadecyl-GPC*
*Plasmalogens Z Score -6 -4 -2 0 2 4 6 Results: Sphingomyelins as new biomarkers for PBD-ZSD Palmitoyl Sphingomyelin Palmitoleoyl Sphingomyelin Oleoyl Sphingomyelin Nervonoyl Sphingomyelin Myristoyl Sphingomyelin Euricoyl Sphingomyelin Eicosenoyl Sphingomyelin Behenoyl Sphingomyelin* Sphingomyelin
-6 -4 -2 0 2 4 6 Z Score NEW DISCOVERIES! Oral Carnitine is converted to Trimethylamine N-oxide (TMAO)
Carnitine TMAO
Gut Microbiota
Backhed F., Nature Medicine (2013) PMID: 23652100 Oral Carnitine is converted to Trimethylamine N-oxide (TMAO)
Carnitine
Gut Microbiota Koeth R.A., et al., Nature Medicine (2013) PMID:23563705 Backhed F., Nature Medicine (2013) PMID: 23652100 Diet & baseline production of TMAO
Koeth R.A., et al., Nature Medicine (2013) PMID: 23563705 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
3-methylhistidine Disorder (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 Michael Wangler
Sarah Elsea Marcus Miller Taraka Donti Paldeep Atwal
Qin Sun Adam Kennedy Leroy Hubert Lisa Emrick Bret Bostwick
What does Global MAPS test for?
• Disorders of amino acid metabolism: • Creatine disorders: plasma/ urine plasma amino acids $220 creatine and guanidinoacetate $280 • Organic acidemias: urine organic acids and • Bile acid disorders: plasma/urine bile acylglycines $500 acids $917 • Purine disorders: urine purine panel $260 • Urea cycle disorders: plasma amino acids & urine orotic acid $300 • Pyrimidine disorders: (urine pyrimidine • Fatty acid oxidation disorders: panel $280 acylcarnitine profile, acylglycines, & urine • Neurotransmitter disorders: plasma organic acids $770 thymidine; urine pyrimidines; plasma/urine • Certain mitochondrial disorders: creatine & guanidinoacetate; csf: – MNGIE: plasma thymidine $200 succinyladenosine, 5HIAA, HVA, 3OMD, lactate, & glucose $1330 (exclusive of LP costs) • Cholesterol Metabolism & PBDs $850
Total cost: > $5000!