13C

13C-SUBSTRATE 13C

FATE ASSOCIATIONS

GUIDED METABOLOMICS

1st Annual Conference on Nutritional Ketosis and Metabolic Therapeutics Tampa, Florida, USA January 28-30, 2016

STABLE ISOTOPE METHODS TO TRACE METABOLIC CHANNELS Aims: • To monitor the fate of specific substrates through biologically relevant enzyme reaction hierarchies •

To determine disease states, drug response and individual variations of metabolism

•

To aid sports medicine

•

….many others

H HO H H H

O C C C C C C H

H OH H OH OH OH

D-glucose

H HO H H H

O C C C C C C H

H OH H OH OH OH

[1,2-13C2]-D-glucose

Mass isotopomer study of the nonoxidative pathways of the pentose cycle with [1,213C ]glucose 2 Am J Physiol. 1998, 274(5 Pt 1):E843-51 Wai-Nang P. Lee et al.

CARBON (12C) • Carbon (12C) nuclei contain six protons and six neutrons • Atomic mass units are 12 (atomic mass unit, Dalton, Da)

CARBON (14C) • Carbon (14C) nuclei contain six protons and eight neutrons • Atomic mass units are 14 (atomic mass unit, Dalton, Da) • Half life is ~5700 years

TRACER CARBONS (13C) • Carbon (13C) nuclei contain six protons and seven neutrons • Atomic mass units are 13 (atomic mass unit, Dalton, Da) • Stable, non-radiating isotopes

13C IS

1.1% IN THE ATMOSPHERE

• Out of one thousand CO2 molecules - 11 are 13C and their concentration remains the same with small variations that depend on temperature, air pressure and altitude •

13CO 2

gas can be collected and stored in containers

13CO •

2

→ METABOLIC TRACERS

13C

containing substrates can be produced by extracting photosynthetic products from e.g. algae that used 13CO2 as the only carbon source

13CO 2

and light

green algae

PHOTOSYNTHESIS

Energy from sunlight

13CO CO

H2O

2

2

Light reactions:

Calvin Cycle

Photosystem II Electron transport chain Photosystem I

O2 Light driven cycle

NADP+ ADP

13C H O n n n

Dark cycle

PHOTOSYNTHESIS AND 13C ENRICHMENT IN PRODUCTS

There are many other 13C labeled products, such as fatty acids and phytochemicals, that can be used as tracers CYS ACP

S S

N=8 acetate units - [U-13C6]-palmitate

H HO H H H

O C C C C C C H

H OH H OH OH OH

[U-13C6]-D-glucose

[U-13CN]-SUBSTRATES (I.) • Non-toxic, non-radiating • No IND needed, cleared by FDA • Molar Mass (C16:0) = 272.43 g·mol−1 • Natural glucose = 256.43 g·mol−1 • 6.2% difference in mass CYS ACP

S S


H HO H H H

O C C C C C C H

H OH H OH OH OH

[U-13CN]-SUBSTRATES (II.) • Chemically identical to natural substrates • Cells, hosts do not recognize it • 1% to 10% enrichment in clinical studies • 0.5 to 1 g/kg (body weight) glucose challenge, $200/g (glucose) CYS ACP

S S


H HO H H H

O C C C C C C H

H OH H OH OH OH

[U-13CN]-SUBSTRATES (III.) Biological Gas-Chromatography and Mass Spectrometry (GC-MS) can find about 400 12C- and 13C- labeled products

CYS ACP

S S

H HO H H H

O C C C C C C H

H OH H OH OH OH

BASIC PRINCIPLES OF GC/MS (II.)

12C

Glucose products fly “fast”

C C C

13C

C C

C

Glucose products fly “slow”

40

DHA-P

4

P

2

6

III.

7

8

9

P P P

CO2

GA-3P IV.

P

P 1,3P-Glyc

Gluc-6P

Fruc-1,6P

39 CO2

21

P

P

PEP

2P-Glyc

3P-Glyc

GLUCONEOGENESIS – SOGC P

P

GA-3P

Oxaloacetate

Acetyl- CoA

Acetyl- CoA

Malate

V.

Low deuterium drinking water

Malate shuttle PENTOSE CYCLE

Fatty acid oxidation (bcarbon)

Citrate

Oxaloacetate

12 III.

Malate

13 20

Fumarate

II.

Low deuterium metabolic water recycling

14

19

Carbons with high deuterium (sugars/amino acids) Carbons with low deuterium (fatty acids from natural fat)

18

Low deuterium carrying fatty acid carbons

Isocitrate

Xylulose-5P Succinate

I.

Succinyl-CoA

15 16 17

CO2

a-ketoglutarate

CO2

KREBS-SZENT-GYÖRGYI CYCLE

Glycolysis Tracer TCA RNA ribose Fatty acid

WT-pFH+262

EV-FH-262

FH-262

FH-268

R2

Correlations

[7] - Glucose tracer consumption (mg/24h)

100.0

128.1

147.5

167.3

1

1

[12] - 13CO2 Glucose oxidation complete (D13C/12C)

100.0

66.3

59.1

76.4

0.3991

-0.6318

[17] - Lactate 13C labeled fraction (Sm)

100.0

107.3

107.8

107.2

0.6793

0.8242

[22] - G6PDH flux NADPH production (m1/m2)

100.0

104.9

105.6

113.6

[22B] - Lactate concentration (peak area)

100.0

123.2

137.0

126.2

[76] - Glutamate 13C labeled fraction (Sm)

100.0

47.9

42.9

34.4

[77] - Glutamate 13C Content (Smn)

100.0

49.9

43.4

33.7

[79] - Glutamate via PDH (m2/Sm)

100.0

82.4

78.6

60.4

[80] - Glutamate via OA recycling (m3/Sm)

100.0

72.9

55.8

58.1

[81] - Glutamate via PC and PDH (m4/Sm)

100.0

226.0

219.8

248.7

[87B] - Glutamate-concentration (peak area)

100.0

74.7

55.6

59.5

[143] - Lignocerate (C24:0) 13C labeled fraction (Sm)

100.0

73.2

64.9

70.1

[144] - Lignocerate (C24:0) 13C Content (Smn)

100.0

67.2

57.2

60.1

[150B] - Lignocerate (C24:0) concentration (peak area)

100.0

176.1

258.8

136.1

100.0

91.8

92.1

74.3

100.0

91.6

92.1

73.7

[296] - RNA-ribose via G6PDH/NADPH (m1/Sm)

100.0

112.2

111.8

140.4

[297] - RNA-ribose via Transketolase (m2/Sm)

100.0

91.2

91.3

74.0

0.8318

-0.9120

[305B] - RNA-ribose concentration (peak area)

100.0

113.2

124.6

116.9

0.6701

0.8186

107 % - 120 %

121 % - 134 %

135 % - 149 %

[294] - RNA-ribose

13

C labeled fraction (Sm)

[295] - RNA-ribose

Percent of Control:

< 64 %

13

C content (Smn)

65 % - 78 %

79 % - 92 %

93 % - 106 %

0.8904 0.9436 Normalization 0.8101 or to0.6562 control the 0.8458 reference -0.9196 time point 0.8720 -0.9338 (100%) 0.9581

-0.9788

0.8740 -0.9349 Color helps to 0.7986 0.8936 monitor flux 0.8614 -0.9281 changes 0.7310

13C

0.7962

-0.8550 -0.8923

normalized 0.1717 0.4143 data to 0.8121 -0.9011 overcome 0.8069 -0.8983 experimental variations 0.7991 0.8939

> 150 %

WT-pFH+262

EV-FH-262

FH-262

FH-268

R2

Correl


100.0

128.1

147.5

167.3

1

1


100.0

104.9

105.6

113.6

0.8904

0.9436


100.0

112.2

111.8

140.4

0.7991

0.8939


100.0

226.0

219.8

248.7

0.7986

0.8936


100.0

107.3

107.8

107.2

0.6793

0.8242


100.0

113.2

124.6

116.9

0.6701

0.8186


100.0

123.2

137.0

126.2

0.6562

0.8101


100.0

176.1

258.8

136.1

0.1717

0.4143


100.0

66.3

59.1

76.4

0.3991

-0.6318


100.0

73.2

64.9

70.1

0.7310

-0.8550


100.0

67.2

57.2

60.1

0.7962

-0.8923

C content (Smn)

100.0

91.6

92.1

73.7

0.8069

-0.8983


100.0

91.8

92.1

74.3

0.8121

-0.9011


100.0

91.2

91.3

74.0

0.8318

-0.9120


100.0

47.9

42.9

34.4

0.8458

-0.9196


100.0

74.7

55.6

59.5

0.8614

-0.9281


100.0

49.9

43.4

33.7

0.8720

-0.9338


100.0

72.9

55.8

58.1

0.8740

-0.9349


100.0

82.4

78.6

60.4

0.9581

-0.9788

107 % - 120 %

121 % - 134 %

135 % - 149 %

> 150 %

[295] - RNA-ribose [294] - RNA-ribose

Percent of Control:

13

< 64 %

13

65 % - 78 %

79 % - 92 %

93 % - 106 %

WT-pFH+262

EV-FH-262

FH-262

FH-268

R2

Correl


100.0

47.9

42.9

34.4

1

1


100.0

49.9

43.4

33.7

0.9986

0.9993


100.0

67.2

57.2

60.1

0.9655

0.9826


100.0

73.2

64.9

70.1

0.9360

0.9675


100.0

72.9

55.8

58.1

0.9227

0.9606


100.0

74.7

55.6

59.5

0.8951

0.9461


100.0

82.4

78.6

60.4

0.8122

0.9012


100.0

66.3

59.1

76.4

0.7196

0.8483


100.0

91.2

91.3

74.0

0.6138

0.7835

100.0

91.8

92.1

74.3

0.5852

0.7650

100.0

91.6

92.1

73.7

0.5787

0.7607


100.0

176.1

258.8

136.1

0.3430

-0.5857


100.0

112.2

111.8

140.4

0.5622

-0.7498


100.0

104.9

105.6

113.6

0.6817

-0.8257


100.0

113.2

124.6

116.9

0.7880

-0.8877


100.0

123.2

137.0

126.2

0.8310

-0.9116


100.0

128.1

147.5

167.3

0.8458

-0.9197


100.0

107.3

107.8

107.2

0.9531

-0.9763


100.0

226.0

219.8

248.7

0.9886

-0.9943

107 % - 120 %

121 % - 134 %

135 % - 149 %

> 150 %

[294] - RNA-ribose

13


[295] - RNA-ribose

Percent of Control:

< 64 %

13

C content (Smn)

65 % - 78 %

79 % - 92 %

93 % - 106 %

o TARGETED 13C FATE ASSOCIATIONS IN DISEASE 167 (±7.05)

279 (±20.70)*

7.2 (±0.16)

5.52 (±0.17)*

22.69 (±0.53)

24.33 (±0.85)

2.49 (±0.005)

2.82 (±0.004)*

307229 (±10561)

387634 (±9403)*

0.82 (±0.25)

0.28 (±0.01)*

0.012 (±0.0005)

0.0053 (±0.0002)*

61.87 (±1.97)

37.36 (±1.06)*

6.13 (±0.13)

3.56 (±0.16)*

5.81 (±0.22)

14.44 (±0.73)*

82860 (±4152)

49335 (±4472)*

31.76 (±0.88)

22.26 (±0.60)*

0.86 (±0.02)

0.51 (±0.014)*

3474 (±104)

4728 (±236)*

14.17 (±0.09)

10.53 (±0.18)*

0.286 (±0.0009)

0.211 (±0.0032)*

42.37 (±1.3)

59.50 (±1.58)*

41.60 (±1.18)

30.76 (±1.04)*

636 (±24)

743 (±12)*

NON-TRACER NON-TARGETED METHODS ARE NON-REPRODUCIBLE

STABLE ISOTOPE METHODS TO TRACE METABOLIC CHANNELS Advantages: • 13C tracer labeled fractions are internal standards •

Product synthesis rates are determined over time and/or a drug dosing regimen (challenge)

STABLE ISOTOPE METHODS TO TRACE METABOLIC CHANNELS ARE HIGHLY REPRODUCIBLE •

13C

mass spectra reflect biology

27

APPLICATIONS IN TARGETED CANCER DRUG DISCOVERY

RESULTS

GLEEVEC INDUCES FATTY ACID OXIDATION, I.E. IMPLEMENTS KETOGENIC DIET, IN LEUKEMIA CELLS

THE JOURNAL OF BIOLOGICAL CHEMISTRY 276(41), 37747-53, 2001

NEJM ARTICLE ABOUT GLEEVEC’S MECHANISMS OF ACTION

NEJM ARTICLE ABOUT GLEEVEC’S MECHANISMS OF ACTION

LETTER TO NEJM EDITOR

LETTER TO NEJM EDITOR

KETOGENIC DIET DEPLETION

- DEUTERIUM

– METABOLIC WATER

Malate

http://high-fat-nutrition.blogspot.com/2012/07/protons-wheres-bias.html

40

DHA-P

4

P

2

6

III.

7

8

9

P P P

CO2

GA-3P IV.

P

P 1,3P-Glyc

Gluc-6P

Fruc-1,6P

39 CO2

21

P

P

PEP

2P-Glyc

3P-Glyc

GLUCONEOGENESIS – SOGC P

P

GA-3P

Oxaloacetate

Acetyl- CoA

Acetyl- CoA

Malate

V.

Low deuterium drinking water

Malate shuttle PENTOSE CYCLE

Fatty acid oxidation (bcarbon)

Citrate

Oxaloacetate

12 III.

Malate

13 20

Fumarate

II.

Low deuterium metabolic water recycling

14

19

Carbons with high deuterium (sugars/amino acids) Carbons with low deuterium (fatty acids from natural fat)

18

Low deuterium carrying fatty acid carbons

Isocitrate

Xylulose-5P Succinate

I.

Succinyl-CoA

15 16 17

CO2

a-ketoglutarate

CO2

KREBS-SZENT-GYÖRGYI CYCLE

1

2

LOW DEUTERIUM CARRYING CARBON

P Gluc

Gluc-6P

22

Transketolase 26 CO2 R-ulose-5P

23 P 6-Phosphogluc

Deoxyribose and DNA strand sugar synthesis

NADPH R-ulose-5P

26

27

4

3 P

5 P

P

DHA-P

PENTOSE CYCLE

GA-3P

GLYCOLYSIS P GA-3P

P Fruc-6P

Fruc-1,6P

APPLICATIONS IN SPORTS MEDICINE

“NO DRUGS IN SPORTS BUT PERFECT MITOCHONDRIAL PRIMING”

- DEUTERIUM DEPLETION – ATP SYNHTESIS

KETOGENIC DIET

Malate

http://high-fat-nutrition.blogspot.com/2012/07/protons-wheres-bias.html

CONCLUSIONS •

Cancer is a deuterium driven metabolic disease

•

Nutritional ketosis using natural fat and fat products control deuterium loading via mitochondrial water recycling into DNA, RNA and biological membranes

•

Oncogenes, oncometabolites and the oncoisotopic role of deuterium need to be explored as drivers of malignant cell transformation

•

Nutritional ketosis needs to be monitored for deuterium enrichment using T1 weighted MR sequence imaging

HEXOSE, PENTOSE, TRIOSE ALDOSE-KETOSE ISOMERIZATION DNA STABILITY

G6PDH NADPH DEUTERIUM DEPLETED WATER ACHIEVES DNA STABILITY VIA GLYCOLYSIS-BOUND LOBRY DE BRUYN TRANSFORMATIONS BY DEPLETING DEUTERIUM2, A KNOWN ONCOISOTOPE, IN PROCESSED DIETARY CARBOHYDRATES

-

CANCER CELL Malate shuttle

Citrate

Fumarate hydratase Hypoxia α-ketoglutarate

DOI: 10.1016/J.MEHY.2015.11.016

LOW DEUTERIUM FATTY ACID Literature: 1DOI: 10.1016/j.mehy.2015.11.016 2http://www.cell.com/molecular-cell/comments/S1097-2765(14)00402-X

OXIDATION AND METABOLIC WATER PRODUCTION ARE DEFECTIVE IN MITOCHONDRIA1

ACKNOWLEDGMENTS Dr. Gábor Somlyai – HYD, LLC Dr. W-N Paul Lee – UCLA Dr. W. Marston Linehan – NCI Dr. Dominic D’Agostino – USF Drs. Howard Katz and Justine Roth – JHU Eszter Boros Agi Hirshberg (pancreatic.org)

HTTPS://WWW.YOUTUBE.COM/USER/FUMARATEHYDRATASELGB