Biology Reference
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in various pathologies, including cancer, to
provide further insights into the dynamics of
cellular metabolism and to pinpoint the precise
deranged pathway. Carbon-13 labeling, which
has been used extensively in metabolic engi-
neering to trace metabolic pathways and
(TCA) is well established. Under isotopic and
metabolic steady-state conditions, glutamate
enrichment is assumed to re
ect the TCA inter-
mediate 2-ketoglutarate and thereby the relative
13
C carbon turnover in the TCA cycle through
the oxidative and anaplerotic pathways.
118
With
judicious
ux
analysis, is ideally suited for this purpose. The
degree of
13
C enrichment depends on the relative
13
C
choice of
labeled substrates,
multiple-bond couplings (
n
J
CC
,n
1) in gluta-
mate C2 and C5 multiplets can provide comple-
mentary TCA metabolic information.
119
Bagga et al. used co-infusion of [U-
13
C
6
]-
glucose and [2-
13
C]-acetate to monitor
13
C
labeling of amino acids to examine region speci
>
flux rates and the labeling pattern of the initial
substrates, which in turn determine the occur-
rence of different
13
C-isotopomers.
The fate of labeled carbons can be followed
using
13
C spectroscopy. In NMR spectroscopy,
but not mass spectroscopy, homonuclear
13
C-
13
C scalar couplings between adjacent
13
C
nuclei allow the determination of positional iso-
topomers. The multiplicity of a particular
13
C
nucleus depends on the degree of labeling of
neighboring carbons. For example, in a three-
carbon segment, if the C
2
carbon is the only
13
C
labeled carbon, it appears as a singlet. If either
the C
1
or C
3
carbon is also
13
C labeled, the C
2
carbon has the following multiplet pattern:
c
neurotoxicity induced by chronic manganese
exposure in a mouse model of Parkinson
s
disease.
120
The incorporation of
13
C labeling
from [U-
13
C
6
]-glucose and [2-
13
C]-acetate in the
brain is shown in
Figure 3
. Neurons and astroglia
incorporate the
13
C label from [U-
13
C
6
]-glucose
intoGlu-C
4,5
, Gln-C
4,5
, andGABA-C
1,2
and astro-
glia incorporate [2-
13
C]-acetate into Glu-C
4
, Gln-
C
4
, and GABA-C
2
.
Figure 4
shows representative
1D
1
H and
13
C spectra of mouse cortical tissue
'
Three carbon fragments
Multiplicity pattern of carbon C
2
12
C
1
-
13
C
2
-
12
C
3
singlet
(1)
13
C
1
-
13
C
2
-
12
C
3
&
12
C
1
-
13
C
2
-
13
C
3
doublet
(2)
13
C
1
-
13
C
2
-
13
C
3
double of doublet or triplet
(3)
extract: indirect carbon detection
1
H[
13
C] in
Figure 4
A, and direct detect carbon spectra in
Figure 4
B
13
C[
1
H]. The spectral expansions
in
Figure 4
B illustrate the multiplet patterns
seen for C
2
of GABBA and C
4
of glutamate and
glutamine.
Instead of 1D
13
C spectra, Yang et al. used 2D
1
H-
13
C HSQC for better spectral dispersion and
were able to quantify 24 metabolites for
final multiplet structure of a carbon NMR
peak is a superposition of the various labeled iso-
topomers present. The relative concentrations of
the isotopomers are determined by the relative
peak areas of individual multiplets deconvoluted
from the parent peak.
117
Statistical calculation of
the
13
C patterns of metabolic intermediates or
end products are used to calculate relative
The
ux
rates.
Central to
ux
analysis through the TCA and pentose phos-
phate pathways using [U-
13
C]-glucose labeling
in human breast cancer cell
flux analysis is the isotope analysis
and a prior knowledge of carbon
flow in the
metabolic pathways. For example, the
lines.
121
Other
flow of
carbon atoms through the tricarboxylic acid cycle
13
C isotopomer
options of 2D experiments for
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