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Mass isotopomers

Mass isotopomers. Molecules with 0 to i heavy atoms Nomenclature: pyruvate: M [1- 13 C]pyruvate: M1 [1,2- 13 C 2 ]pyruvate: M2 [U- 13 C 3 ]pyruvate: M3 Mass isotopomer distribution (MID) assayed by GC-MS or LC-MS.

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Mass isotopomers

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  1. Mass isotopomers • Molecules with 0 to i heavy atoms • Nomenclature: pyruvate: M [1-13C]pyruvate: M1 [1,2-13C2]pyruvate: M2 [U-13C3]pyruvate: M3 • Mass isotopomer distribution (MID) assayed by GC-MS or LC-MS

  2. Metabolomics identifies 2 types of unpredicted correlations • Modulation of one pathway by an intermediate of an apparently unrelated pathway, without transfer of carbon • Unknown transfer of carbon between two pathways: revealed by using a [U-13C]substrate and assaying the Mass Isotopomer Distribution (MID) of the metabolome

  3. Courtesy ofDr. Jeremy K. Nicholson

  4. Metabolomics identifies 2 types of unpredicted correlations • Modulation of one pathway by an intermediate of an apparently unrelated pathway, without transfer of carbon • Unknown transfer of carbon between two pathways: revealed by using a [13C]substrate and assaying the Mass Isotopomer Distribution (MID) of the metabolome

  5. The associationMetabolomics + Isotopomer Analysisyields information on: • Unknown reactions • Links between pathways • Compartmentation of metabolism in intact cells or organs

  6. 3 Strategies for stable isotopic labeling of the metabolome • Focused labeling of one metabolite with its [U-13C]mass isotopomer • Regional labeling with [13C]bicarbonate • General labeling with 2H2O

  7. M5

  8. M1 M5 M5 M1 M1 M4 M1

  9. M1 M M M M M4 M4 M5 M2 M2 M5 M2 M2 M1 M2 M1 M2 M4 M1

  10. M1 M M M M M4 M4 M5 M2 M2 M5 M2 M2 M1 M2 M1 M2 M4 M1

  11. M1 M M M M M4 reactions Unknown enzymes genes M4 M5 M2 M2 M5 M2 M2 M1 M2 M1 M2 M4 M1

  12. Mass isotopomer analysis adds value to metabolomics a.Gluconeogenesis b.Metabolism of odd-chain dicarboxylates

  13. GLUCOSE GLYCOGEN UDPG G1P G6P F6P MERCAPTOPICOLINATE - (Inhibits PEP-carboxykinase) PEP OXALOACETATE LACTATE PYRUVATE Inhibition of gluconeogenesis by mercaptopicolinate

  14. lactate control lactate + MPA 45 40 * 35 30 25 * M1 MPE 20 *# * *# 15 *# * 10 * * 5 * * 0 CIT AKG SUC FUM MAL OAA ASP PEP 2PG 3PG GAP DHAP aGP GLUC -5 5mM lactate perfusions

  15. GLUCOSE GLYCOGEN G6P G1P UDPG NO KNOWN CO2 INCORPORATION IN THIS AREA OF METABOLISM F1,6PP RED ARROWS: CO2 PRODUCTION OR INCORPORATION GAP DHAP αGP FUM 3PG MAL Prop-CoA 2PG SUCC PEP OAA SUCC-CoA MMA-CoA LACT PYR CO2 CIT αKG CO2 ICIT

  16. GLUCOSE 26 = 2 X 13 GLYCOGEN control G6P G1P UDPG F1,6PP 15.8 GAP DHAP αGP 11.9 4.4 FUM 14.5 3PG 22.2 MAL 24.8 Prop-CoA 15.6 2PG 4.5 SUCC 10.4 PEP 17 OAA SUCC-CoA MMA-CoA LACT PYR CO2 CIT 37.4 αKG 16.4 CO2 ICIT

  17. GLUCOSE GLYCOGEN 16 = 2 X 8 + MPA G6P G1P UDPG F1,6PP GAP DHAP αGP 12.6 8.3 0 FUM 5.3 3PG 22.1 MAL Prop-CoA 2PG 21.1 2.5 2.7 SUCC 4.0 PEP OAA 18.7 SUCC-CoA MMA-CoA LACT PYR CO2 CIT 32.5 αKG 7.9 CO2 ICIT

  18. GLUCOSE GLYCOGEN 16 = 2 X 8 + MPA G6P G1P UDPG 13CO2 ?? F1,6PP GAP DHAP αGP 12.6 8.3 0 FUM 5.3 3PG 22.1 MAL Prop-CoA 2PG 21.1 2.5 2.7 SUCC 4.0 PEP OAA 18.7 SUCC-CoA MMA-CoA LACT PYR CO2 CIT 32.5 αKG 7.9 CO2 ICIT

  19. Possible explanations • Unknown carboxylating reaction above PEP • Unknown reactions between a member of the citric acid cycle and trioses-P • Strong labeling heterogeneity of the liver lobule

  20. Implication • Fractional gluconeogenesis may be overestimated by the labeling ratio (glucose)/2(PEP)

  21. Mass isotopomer analysis adds value to metabolomics a. Gluconeogenesis b.Metabolism of odd-chain dicarboxylates

  22. Metabolism of Azelate in Liver • C9n-dicarboxylic acid • Extensively used in dermatology • Initial degradation: peroxisomes? • Final degradation: mitochondria? • Source of peroxisomal malonyl-CoA?

  23. High impact assays • CoA esters (concentration + MID) • Carnitine esters (concentration + MID) • Proxies of the MID of acetyl-CoA pools: total: mitochondrial: acetyl moiety of citrate C1+2 of β-hydroxybutyrate cytosolic: malonyl-CoA peroxisomal: acetate released from liver

  24. AZELATE LYSINE AZELAOYL – CoA C9 α- KETOADIPATE PIMELOYL – CoA C7 ? GLUTARYL - CoA GLUTARYL – CoA C5 CROTONYL - CoA S – BHB - CoA ACETYL - CoA

  25. M9 AZELATE LYSINE AZELAOYL – CoA C9 α- KETOADIPATE PIMELOYL – CoA C7 ? GLUTARYL - CoA GLUTARYL – CoA C5 CROTONYL - CoA S – BHB - CoA ACETYL - CoA

  26. M9 AZELATE LYSINE M9 AZELAOYL – CoA C9 α- KETOADIPATE M7 PIMELOYL – CoA C7 ? GLUTARYL - CoA M5 GLUTARYL – CoA C5 CROTONYL - CoA S – BHB - CoA ACETYL - CoA

  27. (2) M9 AZELATE LYSINE M9 AZELAOYL – CoA C9 α- KETOADIPATE M7 PIMELOYL – CoA C7 ? GLUTARYL - CoA M5 GLUTARYL – CoA C5 CROTONYL - CoA S – BHB - CoA ACETYL - CoA

  28. M9 AZELATE LYSINE M9 AZELAOYL – CoA C9 α- KETOADIPATE M7 PIMELOYL – CoA C7 ? GLUTARYL - CoA M5 GLUTARYL – CoA C5 CROTONYL - CoA M3 MALONYL – CoA + M2 ACETYL-CoA S – BHB - CoA M3 METHYLMALONYL – CoA CAC ACETYL - CoA M3 SUCCINYL-CoA

  29. 9.0 8.0 7.0 6.0 5.0 [MalCoA] (nmols/g.w.w.) 4.0 3.0 2.0 1.0 0.0 0 0.5 1 1.5 2 2.5 M0 azelate (mM)

  30. 70 120 100 60 acetate 80 60 M2 acetate production rate (nmol / min. g.w.w.) 40 50 20 total acetyl CoA 0 0 0.1 0.2 0.3 0.4 0.5 0.6 40 M2 MPE (%) [U-13C9]azelate (mM) 30 acetyl of citrate 20 acetyl-carnitine 10 C1+2 BHB 0 0 0.1 0.2 0.3 0.4 0.5 0.6 [U-13C9]azelate (mM)

  31. 100 M2 malonyl-CoA 2D Graph 1 M2 malonate 80 M3 malonyl-CoA M3 malonate 60 40 20 Mi (%) 4 2 0 0.0 0.1 0.2 0.3 0.4 0.5 [U-13C9]azelate (mM)

  32. 60 50 M3 Malonyl-CoA 40 30 M3 Methylmalonyl-CoA 20 10 M3 Propionyl-CoA 0 0 0.1 0.2 0.3 0.4 0.5 [U-13C9]azelate (mM) M3 enrichment of malonyl-CoA, methylmalonyl-CoA and propionyl-CoA MPEs (%)

  33. M9 AZELATE LYSINE M9 AZELAOYL – CoA C9 GLUCOSE α- KETOADIPATE M7 PIMELOYL – CoA C7 GLUTARYL - CoA M5 GLUTARYL – CoA C5 CROTONYL - CoA PEP M3 MALONYL – CoA + M2 ACETYL-CoA S – BHB - CoA M3 METHYLMALONYL – CoA CAC M Propionyl-CoA ANAPLEROSIS ACETYL - CoA M3 SUCCINYL-CoA

  34. FUM MAL OAA SUCC CIT SUCC-CoA αKG ICIT ANAPLEROTIC SUBSTRATES PYRUVATE ODD-CHAIN FATTY ACIDS PROP-CoA MMA-CoA GLUTAMATE

  35. FUM MAL OAA SUCC CIT SUCC-CoA αKG ICIT ANAPLEROTIC SUBSTRATES PYRUVATE ODD-CHAIN FATTY ACIDS PROP-CoA MMA-CoA GLUTARYL-CoA GLUTAMATE

  36. 35 12 B Succinyl-CoA A α-Ketoglutarate 30 10 [U-13C9]azelate 25 8 [U-13C2]acetate 20 6 Average Carbon labeling (%) Average Carbon labeling (%) 15 4 10 [U-13C9]azelate 2 5 0 0 0 10 20 30 40 50 0 10 20 30 40 50 Acetyl moiety of citrate M2 (%) Acetyl moiety of citrate M2 (%) [U-13C2]acetate

  37. Initial conclusions • Azelate undergoes complete -oxidation to acetyl-CoA + malonyl-CoA in both peroxisomes and mitochondria • Malonyl-CoA derived from azelate appears to be methylated to anaplerotic methylmalonyl-CoA • Looking for methyl donor: methionine, formate, glycine, serine? • Azelate is anaplerotic and gluconeogenic via mitochondrial malonyl-CoA

  38. However, • In livers perfused with unlabeled azelate + either, [methyl-2H3]methionine, [13C]formate, [3-13C]serine, or [2-13C]glycine, MMA-CoA is unlabeled • In livers perfused with [U-13C9]azelate, the free carboxyl of M3 MMA-CoA is unlabeled

  39. Today’s conclusions • M3 malonyl-CoA is NOT methylated to M3 methylmalonyl-CoA • The labeling pattern of MMA-CoA cannot be explained because • It seems to be derived from M3 propionyl-CoA, but • Propionyl-CoA is unlabeled • Unknown reactions link glutaryl-CoA to MMA-CoA

  40. The associationMetabolomics + Isotopomer Analysisyields information on: • Unknown reactions controlled by unknown genes • Links between pathways • Compartmentation of metabolism in intact cells or organs • Applicable to metabolic perturbations associated with neoplasia

  41. Thank you!

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