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domain spectra match with target regions of the
experimental spectrum. Focusing on enhancing
the accuracy for the derived metabolite concen-
trations, three time-zero
1
H-
13
C HSQC series
spectra with incremented repetition times were
used. From this series of spectra, an extrapo-
lated time-zero HSQC spectrum (HSQC
0
) was
obtained.
108,109
Peak intensities in the HSQC
0
spectra represent true concentrations of indi-
vidual metabolites, as they are not in
Ex Vivo
Isotope Labeling
Owing to the extremely high complexity of
biological mixtures, only a small fraction of
metabolites can be accurately analyzed, and the
information derived from such a small number
of metabolite is insuf
cient to gain insights into
altered cellular biochemistry. Targeting different
metabolite classes (acids, amines, etc.) using che-
moselective isotope tags reduces molecular
complexity and improves the detection of low-
concentrationmetabolites by reducing the contri-
bution of less interesting chemical signals. Stable
isotopes such as
13
C and
15
N and abundant heter-
onuclei such as
31
P have been used to tag metab-
olites with speci
uenced
by delays during the pulse sequence that could
cause substantial loss of coherence due to relax-
ation. However, this method is somewhat time
consuming because of the need for multiple
2D spectrum acquisitions. A method that does
not require standard compounds for deter-
mining metabolite concentrations using
1
H-
13
C
HSQC spectra was also reported.
110
This
approach is based on applying a correction
factor to the 2D peak integrals, calculated
from the solution of the Bloch equations and
analysis of product operator formalism utilizing
longitudinal (T
1
) and transverse (T
2
) relaxation
parameters,
1
H-
13
C heteronuclear J-coupling
and the delays used in the 2D pulse sequence.
Applying a correction factor eliminates the
effects of T
1
and T
2
relaxation, heteronuclear
couplings, and experimental parameters and
provides peak integrals that represent true meta-
bolite concentrations.
c functional groups and thereby
signi
cantly enhance the resolution and sensi-
tivity of NMR experiments.
118
e
122
Isotope labeling of metabolites using a
13
C--
acetylation tag results in selective labeling of
amine containing metabolites, and using the
1
H-
13
C HSQC experiment allows the detection of
the
13
C-tagged metabolites with improved resolu-
tion and sensitivity.
118
This labeling approach is
quantitative and the tagging reaction can be
carried out directly in aqueous solution at ambient
temperature. More recently, tagging using for-
mylation using
13
C-formic acid was shown to
improve the detection of amine containingmetab-
olites.
119
The large, 200 Hz, one-bond J-coupling
between the labeled
13
Cand
1
H facilitates ef
cient
transfer of polarization between the two nuclei in
HSQC experiments. Carboxyl functional group
containing metabolites represent another major
class of molecules in biological systems. They can
be chemically tagged with
15
N-ethanolamine and
detected using a 2D
1
H-
15
N HSQC experiment.
120
The enhanced sensitivity and resolution from this
approach enables detection of metabolites at
concentrations as low as a few micromolar, quan-
titatively and reproducibly. Using this approach,
nearly 200 well-resolved signals corresponding
to well over 100 carboxyl-containing metabolites
can be routinely detected in biological mixtures.
A method to detect
Isotope-Labeled NMR
Another approach is the use of isotope
labeling using heteronuclei such as
13
C and
15
N, which provides a number of bene
ts for
quantitative metabolomics. Currently, isotope
labeling is used in two major applications. One
area focuses on tracing metabolic pathways
and
flux analysis using in vivo isotope label-
ing
111
e
117
and the other on increasing the pool
of detected metabolites by enhancing resolution
and sensitivity using ex vivo labeling. Because
the
first area is not used primarily for quantita-
tion, we focus on the second.
lipid metabolites with
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