13C-Substrate Fate Associations - 13C Guided Metabolomics

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DOI: 10.13140/RG.2.1.2581.8643
1st Annual Conference on Nutritional Ketosis and Metabolic Therapeutics, Tampa, Florida, United States of America, DOI:10.13140/RG.2.1.2581.8643
Abstract
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13C-SUBSTRATE FATE ASSOCIATIONS
13C GUIDED METABOLOMICS
1st Annual Conference on Nutritional
Ketosis and Metabolic Therapeutics
Tampa, Florida, USA
January 28-30, 2016
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
STABLE ISOTOPE METHODS TO
TRACE METABOLIC CHANNELS
HO
H
OH C
C
C
C
C
C
OH
O
H
H H
H
H H
OH
OH
D-glucose
HO
H
OH C
C
C
C
C
C
OH
O
H
H H
H
H
OH
OH
[1,2-13C2]-D-glucose
Mass isotopomer study of the
nonoxidative pathways of the
pentose cycle with [1,2-
13C2]glucose
Am J Physiol. 1998, 274(5 Pt
1):E843-51
Wai-Nang P. Lee et al.
H
Carbon (12C) nuclei contain six protons and six neutrons
Atomic mass units are 12 (atomic mass unit, Dalton, Da)
CARBON (12C)
Carbon (14C) nuclei contain six protons and eight neutrons
Atomic mass units are 14 (atomic mass unit, Dalton, Da)
Half life is ~5700 years
CARBON (14C)
Carbon (13C) nuclei contain six protons and seven neutrons
Atomic mass units are 13 (atomic mass unit, Dalton, Da)
Stable, non-radiating isotopes
TRACER CARBONS (13C)
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
13CO2 gas can be collected and stored in containers
13C IS 1.1% IN THE ATMOSPHERE
13C containing substrates can be produced by extracting
photosynthetic products from e.g. algae that used 13CO2 as the only
carbon source
13CO2 and light
green algae
13CO2 METABOLIC TRACERS
H2O
Energy from
sunlight
CO2
Light driven cycle Dark cycle
13CnHnOn
O2
Calvin
Cycle
Light reactions:
Photosystem II
Electron transport chain
Photosystem I
NADP+
ADP
PHOTOSYNTHESIS
13CO2
There are many other
13C labeled products,
such as fatty acids and
phytochemicals, that
can be used as tracers
PHOTOSYNTHESIS AND
13C ENRICHMENT IN PRODUCTS
HO
H
OH C
C
C
C
C
C
OH
O
H
H H
H
H H
OH
OH
[U-13C6]-D-glucose
CYS
ACP S
S
N=8 acetate units - [U-13C6]-palmitate
Non-toxic, non-radiating
No IND needed, cleared by FDA
Molar Mass (C16:0) = 272.43 g·mol1
Natural glucose = 256.43 g·mol1
6.2% difference in mass
[U-13CN]-SUBSTRATES (I.)
HO
H
OH C
C
C
C
C
C
OH
O
H
H H
H
H H
OH
OH
CYS
ACP S
S
N=8 acetate units - [U-13C6]-palmitate
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)
HO
H
OH C
C
C
C
C
C
OH
O
H
H H
H
H H
OH
OH
[U-13CN]-SUBSTRATES (II.)
CYS
ACP S
S
N=8 acetate units - [U-13C6]-palmitate
Biological Gas-Chromatography and
Mass Spectrometry (GC-MS) can find
about 400 12C- and 13C- labeled
products HO
H
OH C
C
C
C
C
C
OH
O
H
H H
H
H H
OH
OH
[U-13CN]-SUBSTRATES (III.)
CYS
ACP S
S
BASIC PRINCIPLES OF GC/MS (II.)
12C Glucose products fly “fast”
13C Glucose products fly “slow”
C
C
C
C
C
C
Fatty acid
oxidation (b-
carbon)
Low
deuterium
carrying fatty
acid carbons
I.
Acetyl- CoA
Acetyl- CoA
Oxaloacetate Citrate
12
Low deuterium
metabolic water
recycling
II.
13
Isocitrate
14
CO2
CO2
a-ketoglutarate
15
16
17
Succinyl-CoA
18
Succinate
19
Fumarate
Malate
20
Malate shuttle
Malate
21
Oxaloacetate
Carbons with high deuterium
(sugars/amino acids)
Carbons with low deuterium
(fatty acids from natural fat) KREBS-SZENT-GYÖRGYI CYCLE
CO2
39
PEP
P
3P-Glyc
9 8
2P-Glyc
P
P
P
P
1,3P-Glyc
7
P
6
GA-3P
P
P
III.
IV.
GLUCONEOGENESIS SOGC
GA-3P
P
DHA-P
4
Fruc-1,6P
Gluc-6P
40
2
CO2
Low
deuterium
drinking
water
V.
III.
PENTOSE CYCLE
P
P
P
Xylulose-5P
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 0.8904 0.9436
[22B] - Lactate concentration (peak area) 100.0 123.2 137.0 126.2 0.6562 0.8101
[76] - Glutamate 13C labeled fraction (Sm) 100.0 47.9 42.9 34.4 0.8458 -0.9196
[77] - Glutamate 13C Content (Smn) 100.0 49.9 43.4 33.7 0.8720 -0.9338
[79] - Glutamate via PDH (m2/Sm) 100.0 82.4 78.6 60.4 0.9581 -0.9788
[80] - Glutamate via OA recycling (m3/Sm) 100.0 72.9 55.8 58.1 0.8740 -0.9349
[81] - Glutamate via PC and PDH (m4/Sm) 100.0 226.0 219.8 248.7 0.7986 0.8936
[87B] - Glutamate-concentration (peak area) 100.0 74.7 55.6 59.5 0.8614 -0.9281
[143] - Lignocerate (C24:0) 13C labeled fraction (Sm) 100.0 73.2 64.9 70.1 0.7310 -0.8550
[144] - Lignocerate (C24:0) 13C Content (Smn) 100.0 67.2 57.2 60.1 0.7962 -0.8923
[150B] - Lignocerate (C24:0) concentration (peak area) 100.0 176.1 258.8 136.1 0.1717 0.4143
[294] - RNA-ribose 13C labeled fraction (Sm) 100.0 91.8 92.1 74.3 0.8121 -0.9011
[295] - RNA-ribose 13C content (Smn) 100.0 91.6 92.1 73.7 0.8069 -0.8983
[296] - RNA-ribose via G6PDH/NADPH (m1/Sm) 100.0 112.2 111.8 140.4 0.7991 0.8939
[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
Percent of Control:
< 64 % 65 % - 78 % 79 % - 92 % 93 % - 106 % 107 % - 120 %
121 % - 134 %
135 % - 149 %
> 150 %
Glycolysis TCA
Fatty acid Tracer
RNA ribose
Normalization
to control or
the reference
time point
(100%)
Color helps to
monitor flux
changes
13C
normalized
data to
overcome
experimental
variations
[7] - Glucose tracer consumption (mg/24h) 100.0 128.1 147.5 167.3 1 1
Percent of Control:
< 64 % 65 % - 78 % 79 % - 92 % 93 % - 106 % 107 % - 120 %
121 % - 134 %
135 % - 149 %
> 150 %
WT-pFH+262 EV-FH-262 FH-262 FH-268 R2 Correl
[22] - G6PDH flux NADPH production (m1/m2) 100.0 104.9 105.6 113.6 0.8904 0.9436
[296] - RNA-ribose via G6PDH/NADPH (m1/Sm) 100.0 112.2 111.8 140.4 0.7991 0.8939
[81] - Glutamate via PC and PDH (m4/Sm) 100.0 226.0 219.8 248.7 0.7986 0.8936
[17] - Lactate 13C labeled fraction (Sm) 100.0 107.3 107.8 107.2 0.6793 0.8242
[305B] - RNA-ribose concentration (peak area) 100.0 113.2 124.6 116.9 0.6701 0.8186
[22B] - Lactate concentration (peak area) 100.0 123.2 137.0 126.2 0.6562 0.8101
[150B] - Lignocerate (C24:0) concentration (peak area) 100.0 176.1 258.8 136.1 0.1717 0.4143
[12] - 13CO2 Glucose oxidation complete (D13C/12C) 100.0 66.3 59.1 76.4 0.3991 -0.6318
[143] - Lignocerate (C24:0) 13C labeled fraction (Sm) 100.0 73.2 64.9 70.1 0.7310 -0.8550
[144] - Lignocerate (C24:0) 13C Content (Smn) 100.0 67.2 57.2 60.1 0.7962 -0.8923
[295] - RNA-ribose 13C content (Smn) 100.0 91.6 92.1 73.7 0.8069 -0.8983
[294] - RNA-ribose 13C labeled fraction (Sm) 100.0 91.8 92.1 74.3 0.8121 -0.9011
[297] - RNA-ribose via Transketolase (m2/Sm) 100.0 91.2 91.3 74.0 0.8318 -0.9120
[76] - Glutamate 13C labeled fraction (Sm) 100.0 47.9 42.9 34.4 0.8458 -0.9196
[87B] - Glutamate-concentration (peak area) 100.0 74.7 55.6 59.5 0.8614 -0.9281
[77] - Glutamate 13C Content (Smn) 100.0 49.9 43.4 33.7 0.8720 -0.9338
[80] - Glutamate via OA recycling (m3/Sm) 100.0 72.9 55.8 58.1 0.8740 -0.9349
[79] - Glutamate via PDH (m2/Sm) 100.0 82.4 78.6 60.4 0.9581 -0.9788
[76] - Glutamate 13C labeled fraction (Sm) 100.0 47.9 42.9 34.4 1 1
Percent of Control:
< 64 % 65 % - 78 % 79 % - 92 % 93 % - 106 % 107 % - 120 %
121 % - 134 %
135 % - 149 %
> 150 %
WT-pFH+262 EV-FH-262 FH-262 FH-268 R2 Correl
[77] - Glutamate 13C Content (Smn) 100.0 49.9 43.4 33.7 0.9986 0.9993
[144] - Lignocerate (C24:0) 13C Content (Smn) 100.0 67.2 57.2 60.1 0.9655 0.9826
[143] - Lignocerate (C24:0) 13C labeled fraction (Sm) 100.0 73.2 64.9 70.1 0.9360 0.9675
[80] - Glutamate via OA recycling (m3/Sm) 100.0 72.9 55.8 58.1 0.9227 0.9606
[87B] - Glutamate-concentration (peak area) 100.0 74.7 55.6 59.5 0.8951 0.9461
[79] - Glutamate via PDH (m2/Sm) 100.0 82.4 78.6 60.4 0.8122 0.9012
[12] - 13CO2 Glucose oxidation complete (D13C/12C) 100.0 66.3 59.1 76.4 0.7196 0.8483
[297] - RNA-ribose via Transketolase (m2/Sm) 100.0 91.2 91.3 74.0 0.6138 0.7835
[294] - RNA-ribose 13C labeled fraction (Sm) 100.0 91.8 92.1 74.3 0.5852 0.7650
[295] - RNA-ribose 13C content (Smn) 100.0 91.6 92.1 73.7 0.5787 0.7607
[150B] - Lignocerate (C24:0) concentration (peak area) 100.0 176.1 258.8 136.1 0.3430 -0.5857
[296] - RNA-ribose via G6PDH/NADPH (m1/Sm) 100.0 112.2 111.8 140.4 0.5622 -0.7498
[22] - G6PDH flux NADPH production (m1/m2) 100.0 104.9 105.6 113.6 0.6817 -0.8257
[305B] - RNA-ribose concentration (peak area) 100.0 113.2 124.6 116.9 0.7880 -0.8877
[22B] - Lactate concentration (peak area) 100.0 123.2 137.0 126.2 0.8310 -0.9116
[7] - Glucose tracer consumption (mg/24h) 100.0 128.1 147.5 167.3 0.8458 -0.9197
[17] - Lactate 13C labeled fraction (Sm) 100.0 107.3 107.8 107.2 0.9531 -0.9763
[81] - Glutamate via PC and PDH (m4/Sm) 100.0 226.0 219.8 248.7 0.9886 -0.9943
167 (±7.05)
7.2 (±0.16)
22.69 (±0.53)
2.49 (±0.005)
307229 (±10561)
0.82 (±0.25)
0.012 (±0.0005)
61.87 (±1.97)
6.13 (±0.13)
5.81 (±0.22)
82860 (±4152)
31.76 (±0.88)
0.86 (±0.02)
3474 (±104)
14.17 (±0.09)
0.286 (±0.0009)
42.37 (±1.3)
41.60 (±1.18)
636 (±24)
279 (±20.70)*
5.52 (±0.17)*
24.33 (±0.85)
2.82 (
±
0.004)*
387634
(±9403)*
0.28 (±0.01)*
0.0053
(±0.0002)*
37.36 (
±
1.06)*
3.56 (±0.16)*
14.44 (
±
0.73)*
49335
(±4472)*
22.26 (
±
0.60)*
0.51 (
±
0.014)*
4728 (±236)*
10.53 (
±
0.18)*
0.211
(±0.0032)*
59.50 (
±
1.58)*
30.76 (
±
1.04)*
743 (±12)*
oTARGETED 13C FATE ASSOCIATIONS IN DISEASE
NON-TRACER NON-TARGETED
METHODS ARE NON-REPRODUCIBLE
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
13C mass spectra reflect biology
STABLE ISOTOPE METHODS TO
TRACE METABOLIC CHANNELS ARE
HIGHLY REPRODUCIBLE
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 GLEEVECS
MECHANISMS OF ACTION
NEJM ARTICLE ABOUT GLEEVECS
MECHANISMS OF ACTION
LETTER TO NEJM EDITOR
LETTER TO NEJM EDITOR
KETOGENIC DIET - DEUTERIUM
DEPLETION METABOLIC WATER
Malate
http://high-fat-nutrition.blogspot.com/2012/07/protons-wheres-bias.html
Fatty acid
oxidation (b-
carbon)
Low
deuterium
carrying fatty
acid carbons
I.
Acetyl- CoA
Acetyl- CoA
Oxaloacetate Citrate
12
Low deuterium
metabolic water
recycling
II.
13
Isocitrate
14
CO2
CO2
a-ketoglutarate
15
16
17
Succinyl-CoA
18
Succinate
19
Fumarate
Malate
20
Malate shuttle
Malate
21
Oxaloacetate
Carbons with high deuterium
(sugars/amino acids)
Carbons with low deuterium
(fatty acids from natural fat) KREBS-SZENT-GYÖRGYI CYCLE
CO2
39
PEP
P
3P-Glyc
9 8
2P-Glyc
P
P
P
P
1,3P-Glyc
7
P
6
GA-3P
P
P
III.
IV.
GLUCONEOGENESIS SOGC
GA-3P
P
DHA-P
4
Fruc-1,6P
Gluc-6P
40
2
CO2
Low
deuterium
drinking
water
V.
III.
PENTOSE CYCLE
P
P
P
Xylulose-5P
P
Gluc
1
P
2
Gluc-6P
22
6-Phosphogluc
P
P
CO2
23
NADPH
R-ulose-5P
26 27
P
Fruc-6P
PENTOSE
CYCLE
P
3
Fruc-1,6P
P
DHA-P
P
GA-3P
5
4
P
GLYCOLYSIS
LOW DEUTERIUM CARRYING CARBON
P
GA-3P
Transketolase
26
R-ulose-5P
Deoxyribose and
DNA strand sugar
synthesis
APPLICATIONS IN SPORTS
MEDICINE
NO DRUGS IN SPORTS BUT PERFECT MITOCHONDRIAL PRIMING
KETOGENIC DIET - DEUTERIUM
DEPLETION ATP SYNHTESIS
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
G6PDH
NADPH
Malate
shuttle Citrate
α-ketoglutarate
DEUTERIUM DEPLETED WATER
ACHIEVES DNA STABILITY VIA
GLYCOLYSIS-BOUND LOBRY DE
BRUYN TRANSFORMATIONS BY
DEPLETING DEUTERIUM2, A
KNOWN ONCOISOTOPE, IN
PROCESSED DIETARY
CARBOHYDRATES
-
DOI: 10.1016/J.MEHY.2015.11.016
Fumarate
hydratase
CANCER CELL
LOW DEUTERIUM FATTY ACID
OXIDATION AND METABOLIC
WATER PRODUCTION ARE
DEFECTIVE IN MITOCHONDRIA1
Literature:
1DOI: 10.1016/j.mehy.2015.11.016
2http://www.cell.com/molecular-cell/comments/S1097-2765(14)00402-X
DNA STABILITY
Hypoxia
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

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