Biology Reference
In-Depth Information
13
(1)
N-acetylation of amino acids and amines
with 1,1-
13
C
2
acetic anhydride in urine and
serum producing a sensitivity gain of
approximately 100-fold in the 1D
13
C
spectra.
105
The N-acetylated amino acid
derivative bearing the isotopically labeled
carbonyl carbons appeared in the small
d
175.7 to 177 spectral region; thus, the use of
the 2D
1
H-
13
C HSQC experiment will be
advantageous.
C:
increased from normal to immortalized to
oncogene-transformed to tumor-derived cells.
112
The methyl protons [-N(CH
3
)
3
] of choline
(
d
3.207 pm), phosphocholine (PC,
d
3.225), and
glycerophosphocholine (GPC,
d
3.234) were
used for quantitation. Since then, studies using
1
H and
31
P have shown that phosphocholine
and total choline are increased in many cancers
and are possible biomarkers.
113
Recent studies
indicate that the GPC/PC ratio may be more
relevant to cancer diagnosis than total choline.
114
However, the narrow chemical shift of the
methyl groups of choline, PC, and GPC are
heavily overlapped in
1
H spectra and are not
differentiable by in vivo techniques in which
spectral resolution is much poorer. With hope
for eventual application in the clinical setting,
two groups recently used spectral editing to
selectively observe the nonoverlapping signals
from the methylene protons of the -CH
2
O group
of PC and GPC. Loening et al. devised a 1D
31
P
edited
1
H NMR experiment and Mao et al.
used
14
N editing in 2D.
115,116
Mao et al. modi
15
(2)
Carboxyl groups tagged with
15
N-ethanolamine followed by 2D
1
H-
15
N
HSQC to detect more than 100 metabolites at
concentrations as lowas a fewmicromolars.
106
N:
31
(3)
Lipids containing aldehyde, carboxyl,
and hydroxyl groups were selectively tagged
with
31
P using the derivatizing agent 2-chloro-
4,4,5,5-tetramethyldioxaphospholane.
107
P:
Targeted Analysis: Metabolite Speci
c
c metabolite is of
interest, 1D
1
H TOCSY can be used to generate
asimpli
In cases where a speci
ed
the one-bond correlation
1
H-
14
N HSQC experi-
ment to a three-bond correlation (HN3BC)
experiment and obtained 2D spectra in several
minutes on ex vivo samples at physiological
temperature (37
C). It is worthwhile to note
that
14
N has a nuclear spin quantum of 1 instead
of the normal spin-½
15
N.
ed spectrum containing just the selec-
tively irradiated peak (the target) and peaks
coupled to it.
108,109
This technique is analogous
to selective or single-ion monitoring in mass
spectrometry and may be the ideal technique
for studies where researchers have prior knowl-
edge of which speci
c metabolites are of
interest. When the target peak is severely over-
lapped with other resonances, Duncan et al.
suggested using the chemical shift selective
Flux Analysis Using
13
C Labeling
The human metabolic network consists of
highly interconnected pathways with regulatory
checks and balances and cross-talks for proper
cellular function. Although pro
filter TOCSY experiment.
110
In another example
of a 1D targeted approach, Tsiafoulis et al.
devised a 1D
1
H pulse sequence to selectively
detect only methyl groups that appear as
a singlet (i.e., the methyl group has no scalar
coupling to other protons in the molecule) in
order to accurately quantitate L-carnitine in
serum samples.
111
Over a decade ago, using breast cancer cell
extracts and
1
H spectroscopy, Aboagye et al.
demonstrated that phosphocholine (PC) and
total
ling steady state
concentration of metabolites can lead to
biomarker discoveries, it does not unequivocally
provide information on the exact pathway per-
turbed. Furthermore, metabolic
ux may change
with no observable change in metabolite concen-
tration. In recent years, there has been a resurgent
interest in using stable isotope labeling to scruti-
nize changes in central carbon metabolism found
choline
concentrations
progressively
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