Biology Reference
In-Depth Information
unknown metabolites as well as quantitation,
from the same measurement. Because the same
nuclei,
1
H for example, are detected with the
same sensitivity, a single internal standard is
suf
spectroscopy) sequence are complicated due to
the
overwhelming macromolecular
signals
from lipids and proteins. It is very dif
cult to
derive quantitative information on metabolites
from such spectra, although spectral
cient to determine the absolute concentra-
tions of all detected metabolites in a single exper-
iment. In addition, the ratios between peaks for
a speci
fitting ap-
proaches can provide limited solutions. Consid-
ering that blood is the most important medium
and is widely used in metabolomics applica-
tions, numerous developments have been made
to avoid the interference of macromolecular
signals. The spin-echo pulse sequence, specifi-
xed and depend on
the number of equivalent nuclei that contribute
to the peak and hence the integrated peak area
for any one isolated peak is suf
c metabolite are
cient to deter-
-
cally its improved version, the Carr-Purcell-
Mieboom-Gill (CPMG) pulse sequence, which
exploits the large difference in the nuclear spin
relaxation properties between metabolites and
macromolecules, is often used to eliminate or
reduce macromolecular signals.
2,67
e
73
Currently,
the CPMG pulse sequence is commonly used for
the analysis of blood serum and plasma samples.
However, caution should be exercised when
using this sequence for quantitative analysis
because the metabolite signals are attenuated
somewhat due to T
2
relaxation. Further, many
physiologically important metabolites including
lactate, ketone bodies, and aromatic amino acids
such as tyrosine, phenylalanine, and histidine
bind to protein molecules in blood serum/
plasma. The
1
H nuclei from such bound metabo-
lites experience a substantial decrease in their
transverse relaxation (T
2
) times and make such
metabolites substantially invisible in the
1
H
NMR spectra
74,75
; due to the line broadening
caused by such binding, some metabolite signals
can even altogether disappear from NMR
spectra. Thus, use of the CPMG sequence can
underestimate concentrations of all endogenous
and exogenous
mine a metabolite
'
s concentration.
Solvent Suppression
A critical requirement for quanti
cation by
NMR is the ef
cient suppression of the water
signal. Owing to its high natural abundance,
sensitivity, and ubiquitous nature,
1
H is the most
preferred nucleus for NMR-based metabolomics.
Bio
uids are aqueous in nature and the concentra-
tion of water in these samples is four orders of
magnitudeormore higher than the typical concen-
trations of metabolites. To date, a large number of
water suppression methods exist, each with its
own advantages and disadvantages. Generally,
these methods use weak radio frequency (RF)
pulses, pulse
field gradients, or their combination
to suppress water signal.
56
e
59
Numerous
improvements have been made that circumvent
many challenges associated with water suppres-
sion and provide spectra without distortions in
phase or peak intensity.
60
e
65
A recently developed
water suppression pulse technique, WET180,
ef
ciently suppresses faraway water that experi-
ences signi
fields relative to
bulk water within the RF coil redgion and enables
sensitive detection of metabolite signals (even
those very close to the water signal).
66
cantly reduced RF
compounds
that bind to
proteins.
74,76
An altogether different approach that
completely separates metabolites and macromo-
lecular signals in blood plasma is shown
in
Figure 3
.
77
This approach utilizes diffusion-
sensitized
1
H NMR spectroscopy and exploits
a large difference in the translational diffusion
coef
Suppression of Macromolecular Signals
NMR spectra of bio
uids such as blood serum
and plasma obtained using the single pulse or
1D NOESY
(nuclear Overhauser
effect
cients between blood plasma metabolites
Search WWH ::
Custom Search