Biology Reference
In-Depth Information
and macromolecules to separate signals from the
two groups. Two separate NMR spectra are
obtained: one that is minimally affected by relax-
ation and diffusion and another in which all
metabolite signals are removed based on their
faster diffusion, which causes the NMR signal
to attenuate. Subtraction of the latter spectrum
from the
mixtures. The use of a coaxial capillary tube con-
taining a solution of TSP or DSS in deuterium
oxide (D
2
O) offers a number of advantages.
The reference compound does not interact with
the sample matrix; it serves as a chemical shift
as well as a quantitative reference, and the D
2
O
solvent
field-frequency locking
solvent. Further, the same capillary tube can be
used for all samples, which adds to minimizing
the quantitation errors. Nevertheless, this
approach adds some additional steps and is
hard to automate.
Somewhat recently, considering that water is
highly concentrated in biological mixtures,
a method that uses water itself as a concentration
reference was proposed.
83
Here, the very large
solvent (water) signal is obtained in a separate
experiment with reduced receiver gain. The inte-
gral of the solvent signal is then compared with
those of metabolites using the two spectra to
obtain absolute concentrations of the metabolites
of interest. It was shown that quantitation of
metabolites can be made with errors less than
2% over a wide range of concentrations. This
method does not require additional compounds
for concentration referencing and is indifferent
to probe tuning. For accurate quantitation,
however, it assumes a linear response to radio-
frequency pulse length and transmitter power,
and receiver gain settings, which is generally
true. Several following reports discuss the in
serves as a
first provides a spectrum for blood
plasma metabolites, which is devoid of broad
peaks as well as baseline distortions frommacro-
molecules. Another important method for
macromolecular signal suppression includes
physically removing proteins from the biological
mixtures. There are numerous ways for such
removal of proteins from blood plasma and
serum including precipitation by adding organic
solvents, salts, or altering pH, or by ultra
ltra-
tion.
78
The performance of such deproteinization
methods is shown to vary signi
cantly, and
hence caution should be exercised while opti-
mizing the deproteinization protocol.
79
Owing
to their superior performance, deproteinization
by ultracentrifugation using 3kDa cutoff
filters,
for example,
79,80
and precipitation using meth-
anol solvent
81
are more commonly used for
quantitative applications.
Quantitative Referencing
Adding a known amount of TSP [sodium
d
4
-3- (trimethylsilyl)-propionate] or DSS (sodium
d
6
-2, 2-dimethyl-2-silapentane-5-sulfonate) can
provide a signal that can be used as both a quan-
titative and chemical shift reference. However,
the binding of these standards with macromole-
cules such as proteins and their sensitivity to
sample conditions such as pH can lead to severe
signal attenuation or peak shifting; hence, addi-
tion of these compounds directly into biological
mixtures is generally not preferred. To circum-
vent this problem, formate was explored as
a quantitative reference, which reportedly does
not interact with macromolecules.
82
Its utility
as a reference for quantitation is limited because
formate is present generally in most biological
u-
ence of such parameters on the accuracy of
metabolite quantitation.
84
e
87
A method known
as ERETIC (Electronic REference To access In
vivo Concentrations) that does not require either
a reference compound or a solvent signal for
quantitation has also been proposed.
88
It
involves synthesis of a reference signal by an
electronic device, and its position in the spec-
trum can be conveniently chosen so as to not
interfere with metabolite signals. For accurate
quantitation, however, the ERETIC method
requires occasional calibration using a standard
compound. Although this approach is very
simple, the results are reliable only when the
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