# Presentation: 050887

## 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

## 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

## Courtesy of  Dr. Jeremy K. Nicholson

**Courtesy of****  ****Dr. Jeremy K. Nicholson**

## 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

**Unknown transfer of carbon between two pathways: revealed by using a [13C]substrate and assaying the Mass Isotopomer Distribution (MID) of the metabolome**

**Metabolomics identifies 2 types of unpredicted correlations**

## Unknown reactions
Links between pathways
Compartmentation of metabolism               in intact cells or organs

- Links between pathways
- Compartmentation of metabolism               in intact cells or organs

- The association** ****Metabolomics + Isotopomer Analysis**** **yields information on:

## 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

## Slide 7

## M5

**M5**

## M5

**M5**

**M5**

**M1**

**M4**

**M1**

**M1**

## M5

**M5**

**M5**

**M1**

**M4**

**M1**

**M1**

**M4**

**M4**

**M2**

**M2**

**M2**

**M2**

**M2**

**M2**

**M**

**M**

**M**

**M**

## M5

**M5**

**M5**

**M1**

**M4**

**M1**

**M1**

**M4**

**M4**

**M2**

**M2**

**M2**

**M2**

**M2**

**M2**

**M**

**M**

**M**

**M**

## 	         reactions

**M5**

**M5**

**M1**

**M4**

**M1**

**M1**

**M4**

**M4**

**M2**

**M2**

**M2**

**M2**

**M2**

**M2**

**M**

**M**

**M**

**M**

**	****         ****reactions**
**Unknown    enzymes**
**	****         ****genes**

## Mass isotopomer analysis adds value to metabolomics

**a.** **Gluconeogenesis**
- b. Metabolism of odd-chain dicarboxylates

## Inhibition of gluconeogenesis by mercaptopicolinate

## 5mM lactate perfusions

**5mM lactate perfusions**

## F1,6PP

**DHAP**

**α****G****P**

**G6P**

**GLUCOSE**

**UDPG**

**G1P**

**GLYCOGEN**

**PYR**

**LACT**

**PEP**

**3PG**

**GAP**

**2PG**

**Prop-CoA**

**MMA-CoA**

**CO****2**

**CO****2**

**FUM**

**ICIT**

**CIT**

**α****KG**

**SUCC-CoA**

**OAA**

**MAL**

**SUCC**

**NO KNOWN CO****2**** INCORPORATION IN THIS AREA OF METABOLISM**

**RED ARROWS: CO****2**** PRODUCTION OR INCORPORATION**

## control

**DHAP**

**α****G****P**

**G6P**

**GLUCOSE**

**UDPG**

**G1P**

**GLYCOGEN**

**PYR**

**LACT**

**PEP**

**3PG**

**GAP**

**2PG**

**MMA-CoA**

**CO****2**

**CO****2**

**FUM**

**ICIT**

**CIT**

**α****KG**

**SUCC-CoA**

**OAA**

**MAL**

**SUCC**

**10.4**

**15.6**

**14.5**

**15.8**

**11.9**

**4.4**

**26 = 2 X 13**

**control**

**22.2**

**37.4**

**16.4**

**4.5**

**17**

**24.8**

**Prop-CoA**

## + MPA

**DHAP**

**α****G****P**

**G6P**

**GLUCOSE**

**UDPG**

**G1P**

**GLYCOGEN**

**PYR**

**LACT**

**PEP**

**3PG**

**GAP**

**2PG**

**Prop-CoA**

**MMA-CoA**

**CO****2**

**CO****2**

**FUM**

**ICIT**

**CIT**

**α****KG**

**SUCC-CoA**

**OAA**

**MAL**

**SUCC**

**4.0**

**2.5**

**5.3**

**12.6**

**8.3**

**0**

**+ MPA**

**16 = 2 ****X ****8**

**22.1**

**21.1**

**18.7**

**32.5**

**7.9**

**2.7**

## + MPA

**DHAP**

**α****G****P**

**G6P**

**GLUCOSE**

**UDPG**

**G1P**

**GLYCOGEN**

**PYR**

**LACT**

**PEP**

**3PG**

**GAP**

**2PG**

**MMA-CoA**

**CO****2**

**CO****2**

**FUM**

**ICIT**

**CIT**

**α****KG**

**SUCC-CoA**

**OAA**

**MAL**

**SUCC**

**4.0**

**2.5**

**5.3**

**12.6**

**8.3**

**0**

**13****CO****2 ****??**

**+ MPA**

**16 = 2 ****X ****8**

**22.1**

**21.1**

**18.7**

**32.5**

**7.9**

**2.7**

**Prop-CoA**

## 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

## Implication

- Fractional gluconeogenesis may be overestimated by the labeling ratio (glucose)/2(PEP)

## Slide 23

## Metabolism of Azelate in Liver

- C9 *n*-dicarboxylic acid
- Extensively used in dermatology
- Initial degradation: peroxisomes?
- Final degradation: mitochondria?
- Source of peroxisomal malonyl-CoA?

## 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

## Slide 26

## Slide 27

## Slide 28

## (2)

## Slide 31

## [MalCoA] (nmols/g.w.w.)

**M0 azelate (mM)**

**0.0**

**1.0**

**2.0**

**3.0**

**4.0**

**5.0**

**6.0**

**7.0**

**8.0**

**9.0**

**0**

**0.5**

**1**

**1.5**

**2**

**2.5**

## Slide 33

## Slide 35

## M3 enrichment of malonyl-CoA, 

- methylmalonyl-CoA and propionyl-CoA

**MPEs (%)**

## Slide 37

## ANAPLEROTIC SUBSTRATES

**PYRUVATE**

**GLUTAMATE**

**MMA-CoA**

**PROP-CoA**

**ODD-CHAIN FATTY ACIDS**

**ANAPLEROTIC SUBSTRATES**

## ANAPLEROTIC SUBSTRATES

**PYRUVATE**

**GLUTAMATE**

**MMA-CoA**

**PROP-CoA**

**ODD-CHAIN FATTY ACIDS**

**GLUTARYL-CoA**

**ANAPLEROTIC SUBSTRATES**

## Slide 40

## 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

## 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

## 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**

## The association Metabolomics + Isotopomer Analysis yields 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

## Thank you!