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SUPPLEMENTARY MATERIALS
to
Profiling of Central Metabolism in Human Cancer Cells by Twodimensional NMR, GC-MS Analysis, and Isotopomer Modeling
Chen Yang1§, Adam D. Richardson2§, Andrei Osterman1* and Jeffrey W. Smith2,3*
Inflammatory and Infectious Disease Center1, The Cancer Center2, and Proteomics
Center3, The Burnham Institute for Medical Research, 10901 North Torrey Pines Road,
La Jolla, California 92037
1
Table A1. Central metabolic network in human cells with the stoichiometric reactions and the
corresponding carbon atom transitions a
Enzyme
Glycolysis
Hexokinase
Gene b Reaction No.
in Fig.1 c
HK
r1
Glucose phosphate isomerase
GPI
r2
Phosphofructose kinase
PFK
r3
Fructose-1,6-bisphosphate
aldolase
Triosephosphate isomerase
ALDO
r4
TPI
r5
Glyceraldehyde-3-phosphate
dehydrogenase
GAPDH r6
Phosphoglycerate kinase
PGK
Phosphoglycerate mutase
PGAM r7
Enolase
ENO
r7
Pyruvate kinase
PK
r8
Lactate dehydrogenase
LDH
r9
G6PD
r10
PGLS
r10
PGD
r11
RPIA
r12
RPE
r13
TKT
r14
TALDO1
r15
TKT
r16
Pentose phosphate pathway
Glucose-6-phosphate
dehydrogenase
6-Phosphogluconolactonase
6-Phosphogluconate
dehydrogenase
Ribose-5-phosphate
isomerase
Ribulose-5-phosphate
epimerase
Transketolase 1
Transaldolase
Transaldolase 2
r6
TCA cycle and anaplerotic pathway
Pyruvate dehydrogenase PDHA, PDHB r17
DLAT, DLD
Citrate synthase
CS
r18
Aconitase
ACO
r19
Isocitrate dehydrogenase
IDH
r20
Stoichiometric reaction
C atom transition d
GLC + ATP => G6P + ADP
abcdef
=> abcdef
G6P <=> F6P
abcdef <=> abcdef
F6P + ATP => FBP + ADP
abcdef
=> abcdef
FBP <=> DHAP + GAP
abcdef <=> abc + def
DHAP <=> GAP
abc
<=> cba
GAP + PI + NAD <=> 13BPG + NADH
abc
<=> abc
13BPG + ADP <=> PGA + ATP
abc
<=> abc
PGA <=> 2PG
abc <=> abc
2PG <=> PEP
abc <=> abc
PEP + ADP => PYR + ATP
abc
=> abc
PYR + NADH <=> LAC + NAD
abc
<=> abc
G6P + NADP => 6PGL + NADPH
abcdef
=> abcdef
6PGL => 6PG
abcdef => abcdef
6PG + NADP => Ru5P + CO2 + NADPH
abcdef
=> bcdef + a
Ru5P <=> R5P
abcde <=> abcde
Ru5P <=> X5P
abcde <=> abcde
R5P + X5P <=> S7P + GAP
abcde + fghij <=>fgabcde + hij
S7P + GAP <=> F6P + E4P
abcdefg + hij <=>abchij + defg
X5P + E4P <=> F6P + GAP
abcde + fghi <=>abfghi + cde
PYR + CoA + NAD => ACoA + CO2 + NADH
abc
=> bc + a
OAA + ACoA => CIT + CoA
abcd + ef
=> dcbafe
CIT <=> ICT
abcdef <=> abcdef
ICT + NAD(P) => AKG + CO2 + NAD(P)H
abcdef
=> abcef + d
2
2-Ketoglutarate dehydrogenase OGDH r21
DLST, DLD
Succinate thiokinase
SUCLG r21
Succinate
dehydrogenase
Fumarate hydratase
SDHA, SDHB, r22
SDHC, SDHD
FH
r23
Malate dehydrogenase
MDH
r24
Malic enzyme
ME
r25
Pyruvate carboxylase
PC
r26
Amino acid synthesis
Phosphoglycerate
PHGDH
dehydrogenase
Phosphoserine
PSAT1
aminotransferase
Phosphoserine
PSPH
phosphotase
Serine
SHMT
hydroxymethyltransferase
Cystathionine β-synthase
CBS
r27
r27
r27
r28
r29
Cystathionase
CTH
r29
Glutamic-pyruvic
transaminase
Glutamate dehydrogenase
GPT
r30
GLUD
r31
Glutamate transport
−
r32
Glutamine synthetase
GLNS
r33
Pyrroline-5-carboxylate
synthase
Pyrroline-5-carboxylate
reductase
Glutamate-cysteine
ligase
Glutathione synthetase
P5CS
r34
PYCR
r34
GCLC
r35
GSS
r35
ACLY
r36
Fatty acid synthesis
ATP citrate lyase
AKG + NAD + CoA => SUCCoA + CO2 + NADH
abcde
=> bcef + a
SUCCoA + GDP + PI <=> SUC + CoA + GTP
abcd
<=> abcd
SUC + UQN <=> FUM + UQL
abcd
<=> abcd
FUM <=> MAL
abcd <=> abcd
MAL + NAD <=> OAA + NADH
abcd
<=> abcd
MAL + NAD(P) <=> PYR + CO2 + NAD(P)H
abcd
<=> abc + d
PYR + CO2 + ATP => OAA + PI + ADP
abc + d
=> abcd
PGA + NAD <=> PHP + NADH
abc
<=> abc
PHP + Glu <=> PSer + AKG
abc
<=> abc
PSer => Ser + PI
abc => abc
Ser + THF <=> Gly + METTHF
abc
<=> ab + c
Ser + HCys => CTH
abc + defg => abcgfed
CTH => Cys + AKB + NH3
abcdefg => abc + gfed
PYR + Glu <=> Ala + AKG
abc
<=> abc
AKG + NH3 + NAD(P)H <=> Glu + NAD(P)
abcde
<=> abcde
GLU* => GLU
abcde => abcde
Glu + NH3 + ATP <=> Gln + ADP + PI
abcde
<=> abcde
Glu + ATP + NADPH => P5C + NADP + ADP+ PI
abcde
=> abcde
P5C + NAD(P)H => Pro + NAD(P)
abcde
=> abcde
Glu + Cys + ATP => Glu-Cys + ADP + PI
abcde + fgh
=> abcdehgf
Glu-Cys + Gly + ATP => GSH + ADP + PI
abcdefgh + ij
=> abcdefghji
CIT + ATP + CoA => OAA + ACoA + ADP + PI
abcdef
=> dcba + fe
Acetyl-CoA carboxylase
ACAC r37
ACoA + CO2 + ATP => MCoA + ADP + PI
ab + c
=> abc
Fatty acid
FASN
r38
ACoA + 7 MCoA + 14 NADPH => C16:0 + 7 CO2 + 8 CoA + 14 NADP
synthase
ab + cde
=> cdcdcdcdcdcdcdab
Fatty acyl-CoA elongase ELO,
r39
C16:0-CoA + ACoA + 2 NADPH => C18:0-CoA + 2 NADP
KAR, TER
abcdefghijklmnop + qr
=> abcdefghijklmnopqr
Stearoyl-CoA desaturase SCD
r40
C18:0-CoA + NADH + O2 => C18:1-CoA + NAD
abcdefghijklmnopqr
=> abcdefghijklmnopqr
3
a
The metabolic pathways were inferred from current literature and data deposited in databases.
Abbreviations: 13BPG, 1,3-bisphosphoglycerate; 2PG, 2-phosphoglycerate; 6PG, 6-phosphogluconate;
6PGL, 6-phosphoglucono-δ-lactone; ACoA, acetyl-coenzyme A; AKB, α-ketobutyrate; AKG, αketoglutarate; CTH, cystathionine; DHAP, dihydroxyacetone phosphate; E4P, erythrose-4-phosphate; F6P,
fructose-6-phosphate; FBP, fructose-1,6-bisphosphate; FUM, fumarate; G6P, glucose-6-phosphate; GAP,
glyceraldehyde-3-phosphate; GLC, glucose; HCys, homocysteine; GSH, glutathione; ICT, isocitrate; LAC,
lactate; MAL, malate; MCoA, malonyl-coenzyme A; METTHF, N5,N10-methylene-tetrahydrofolate; OAA,
oxaloacetate; P5C, Δ1-pyrroline-5-carboxylate; PEP, phosphoenolpyruvate; PGA, 3-phosphoglycerate; PHP,
3-phosphohydroxypyruvate; PSer, 3-phosphoserine; PYR, pyruvate; R5P, ribose-5-phosphate; Ru5P,
ribulose-5-phosphate; S7P, seduheptulose-7- phosphate; SUC, succinate; SUCCoA, succinyl-coenzyme A;
THF, tetrahydrofolate; UQL, ubiquinol; UQN, ubiquinone; X5P, xylulose-5-phosphate;
b
Because our method can not differentiate between isoenzymes, an overall gene name is given for the
isoenzymes.
c
The number of reactions correspond to that in Fig. 1. Some reactions are lumped for simplicity in Fig. 1.
For example, the conversion of glyceraldehyde-3-phosphate to 3-phosphoglycerate by glyceraldehydes-3phosphate dehydrogenase and phosphoglycerate kinase is lumped as r 6 in Fig. 1.
d
For each reaction, the corresponding carbon atom transitions are given. For example, for transaldolase in
the pentose phosphate pathway (r15), the first carbon of GAP (denoted by h) becomes the fourth carbon of
F6P.
4
Table A2. 1H and 13C chemical shifts of resonances from different metabolites present in the breast cancer
cells a
Serial
Metabolites
no.
Carbon
1
H multiplet
13
C multiplet
1
H chemical
13
C chemical
b
c
shift (ppm)
shift (ppm)
C2
q
m
3.79
53.24
C3
d
m
1.49
18.89
position
Amino acids
1
Alanine
2
Arginine
C5
t
s
3.26
43.26
3
GSH (Glu)
C3
m
m
2.18
28.91
C4
t
m
2.57
34.14
C2
t
s
4.58
58.40
C3
m
s
2.97
28.25
GSH (Gly)
C2
s
s/m
3.79
46.10
Glutamate
C2
t
m
3.79
56.90
C3
m
m
2.07, 2.13
29.58
C4
t
m
2.36
36.22
GSH (Cys)
4
5
Glutamine
C4
m
m
2.46
33.56
6
Glycine
C2
s
s/m
3.57
44.20
7
Isoleucine
C4-H3
d
s
1.02
17.39
C5-H3
t
s
0.95
13.83
C5
d
s
0.98
24.75
C5′
d
s
0.97
23.67
C5
m
s
1.74
29.12
C6
t
s
3.02
41.95
C4
m
s/m
2.02
26.49
C5
t, t
s/m
3.37, 3.44
48.85
C4
d
s
1.05
20.68
C4′
d
s
1.00
19.39
8
9
10
11
Leucine
Lysine
Proline
Valine
5
Organic acids
12
13
Lactate
CH
q
m
4.12
71.22
CH3
d
m
1.33
22.78
Succinate
CH2-CH2
s
m
2.42
36.90
m-Inositol
C1H, C3H
q
s
3.55
73.87
C2H
t
s
4.07
74.92
C4H, C6H
q
s
3.63
75.15
C5H
t
s
3.29
77.09
CH3
d
m
2.01
24.62
CH3
d
m
2.08
24.83
CH2OH
m
s
4.07
58.35
NCH2
m
s
3.52
70.17
CH2-PO3-
m
s
4.18
60.79
NCH2
m
s
3.60
69.23
N(CH3)3
s
s
3.23
56.75
CH2-PO3-
m
s
4.33
62.17
NCH2
m
s
3.68
68.74
CH2-O-P
m
m
3.89, 3.95
69.25
CHOH
m
m
3.91
73.38
CH2OH
m
m
3.62, 3.68
64.76
C1H
d
m
6.00
91.14
C2H
t
m
4.39
76.49
Sugars
14
15
GlcNAc
/ GalNAc
16
UDP-GlcNAc
/UDP-GalNAc
Membrane components
17
18
19
Choline
Phosphocholine
Glycerophosphocholine
Nucleotides
20
UTP / UDP
6
C3H
t
m
4.38
72.39
C4H
t
m
4.30
85.96
C5H2
m
m
4.24
67.89
CH2
s
s
3.94
56.63
CH3
s
s
3.05
39.69
N-CH2
t
s
3.44
38.15
S-CH2
t
s
3.28
50.23
Other compounds
21
Creatine
22
Taurine
a
Abbreviations: GSH, glutathione; GlcNAc, N-acetylglucosamine; GalNAc, N-acetylgalactosamine; s,
singlet; d, doublet; t, triplet; q, quartet; m, multiplet.
b1
H multiplets were obtained from 1H-NMR spectra. These data have been reported by Sitter et al (Sitter et
al. 2002).
c 13
C multiplets were obtained based on [U-13C]glucose labeling and 2D [13C, 1H] HSQC spectroscopy.
7
Table A3. Equations used for calculation of metabolic fluxes or flux ratios a
Metabolic flux or flux ratio
Equation
Pyruvate from PPP b
5/2 × f(2)(Ala-C2)
(A1)
Ribose from oxidative PPP b
f(3)(UTP/UDP-C2)
(A2)
Ribose from non-oxidative
f(1) + f(2) (UTP/UDP-C2)
(A3)
(A4)
to TCA cycle
d (Glu  C3)
d * (Glu  C4)  dd (Glu  C4)
Malic Enzyme b
f(2*)(Ala-C2)
(A5)
PPP
b
Contribution of anaplerosis
(1  Pn ) 2 
s 


d 
(1  Pn )  Pn 
Pn  (  

)
d *
(1  Pn )  Pn 


 

dd  G ln C 4  Pn 2
Glutamine (or glutamate)
from glucose
(A6)
(1  Pn ) 2 
s 
s 
s 


d 
d 
d 
(1  Pn )  Pn 
PGlu(GSH) C 4  (  
 
)  Pn  (  

)
d *
d *
d *
(1  Pn )  Pn 


 
 
 

dd  Glu(GSH) C 4 dd  G ln C 4
dd  G ln C 4  Pn 2
Glycine from glucose
1  Pn 
s 
Pn  (  

)
d  GlyC 2  Pn 
1  Pn 
s  d 
s 
s 
PAlaC 2  ( 
 
)  Pn  (  

)

d *  dd  AlaC 2 d  GlyC 2
d  GlyC 2  Pn 
(1  Pn ) 2

s 




Pn  ( d 
 2  (1  Pn )  Pn  )


2
t 
Pro C 4  Pn

Proline from glucose
s

PGlu C 4  ( d  d *
dd

De novo synthesis of
palmitate (C16:0)
De novo synthesis of
stearate (C18:0) and oleate
Glu C 4
s 
 d 
t 
Pro C 4
s 
)  Pn  ( d 
t 
(A7)
(A8)
(1  Pn )



 2  (1  Pn )  Pn  )
 2

Pro C 4  Pn

2
m2 / (8 · p · q7) or m4 / (28 · p2 · q6)
(A9)
p = (m4/m2) / (3.5 + (m4/m2)) or (m6/m4) / (2 + (m6/m4)), p + q =1
m2 / (9 · p · q8) or m4 / (36 · p2 · q7)
(A10)
p = (m4/m2) / (4 + (m4/m2)) or (m6/m4) / (7/3 + (m6/m4)), p + q =1
(C18:1)
a
See the text for derivation of equations. s, singlet; d, doublet split by a small coupling constant; d*, doublet
with a larger coupling constant; t, triplet; dd, doublet of doublets; Pn, 13C natural abundance (0.012); m,
mass isotopomer distribution.
8
b
The abundances of intact carbon fragments originating from a single glucose source molecule (f values)
were calculated from the observed relative intensities of 13C multiplets (i.e. s, d, t, or dd) as described by
(Szyperski 1995). The denotation of f has been described in (Szyperski 1995). Briefly, f (1) represents the
fraction of molecules in which the observed carbon atom and its neighboring carbons originate from
different source molecules of glucose, and f (2) the fraction of molecules in which the observed carbon atom
and at least one neighboring carbon originate from the same source molecule. For a central carbon in a C3
fragment that exhibits different 13C-13C scalar coupling constants with the two attached carbons, f (2)
represents the fraction of molecules for which the central carbon and the carbon with the smaller coupling
come from the same source molecule, while f (2*) is used if the carbon with the larger coupling comes from
the same source molecule as the observed carbon. f (3) denotes the fraction of molecules in which the
observed carbon atom and both neighbors in the C3 fragment originate from the same glucose molecule.
9
Figure Legends
13
1
Fig.A1. A typical two-dimensional [ C, H] HSQC spectrum of the metabolites extracted
from MCF-7 breast cancer cells. Abbreviations: Lac, lactate; Suc, succinate; mI,
m-inositol; FC, choline; PC, phosphocholine; GPC, glycerophosphocholine; Cr,
creatine; Tau, taurine.
10
GlcNAc/
UDPGlcNAc/
UDPGalNAc-CH3 GalNAc-CH3
Cys(GSH)-C3
Glu(GSH)-C3
Glu(GSH)-C4
Tau-NCH2
Pro-C5
Cr-CH2
Lys-C5
Glu-C4
Lys-C6
Tau-SCH2
13C
Ala-C2
FC-CH2OH
Pro-C4
Lac-C3
Glu-C3
Suc-C2/C3
Gly(GSH)-C2
Cys(GSH)-C2
Val-C4
Cr-CH3
Arg-C5
Gly-C2
Ala-C3
Glu-C2
PC/GPC/FC-N(CH3)3
PC-CH2PO3UTP/UDP-C5
PC-NCH2
mI-C1/C3
Lac-C2
mI-C2
mI-C4/C6
mI-C5
1H
Fig. A1
C. Yang et al.
11