Biology Reference
In-Depth Information
concentrations of isotope-labeled metabolites in
algal or bacterial extracts, however, is needed,
utilizing unlabeled standard mixtures of metabo-
lites, and one needs to be careful about SILIS
sample stability. Nevertheless, using this in vivo
e
derived SILIS compound approach, more than
90% of measured metabolites were shown to
exhibit CVs of less than 15%.
33
This method has
fostered increased interest for determining abso-
lute concentrations of cellular metabolites,
18,40
e
45
and it will be interesting to see whether it
promises a robust method for mass spectrome-
try
e
based quantitative metabolomics using
commonly used biological samples such as blood,
urine, and tissue. Detailed step-by-step experi-
mental protocols for quantitation of intracellular
metabolites and measurement of cellular
FIGURE 2
Distribution of CV values for metabolites in rat
plasma obtained using a small set of representative internal
standards (SILIS).
30
ed nucleosides in biological samples.
35,36
The use of structural analogues or a small
number of representative SILIS compounds can
be cost-effective in targeted metabolite quantita-
tion. However, spiking with a SILIS for each
metabolite provides more reliable results (intra-
and interday CVs
modi
uxes
have been described using examples of Escherichia
coli and primary human
fibroblasts fed with
U-
13
C-labeled carbon sources and labeledmetabo-
lite detection using LC-MS/MS.
46,47
In the previously described quantitation
methods using a limited number of labeled
internal standards, external calibration curves
are usually used to calculate the metabolic
concentrations. These calibration curves are
obtained from calibrant samples that were spiked
with labeled internal standards. However, some
studies utilized external calibration curves
without spiking the calibrants with labeled
internal standards, and the metabolic concentra-
tions were determined from a plot of the MS
peak intensity versus standard metabolite
concentration. The advantage of this approach is
that the calibrant samples do not require spiking
with labeled standards. The disadvantage of this
approach is that the calibration curves are based
on the series of standard dilutions with signifi-
10%), as only the labeled
counterpart can truly mimic the response of an
individual metabolite as compared to a structur-
ally similar internal standard or representative
labeled metabolite.
37,38
An approach that does not depend on
the commercially available isotope-labeled
compounds and eliminates a major drawback of
LC-MS-based metabolite quanti
<
cation utilizes
isotope-labeled metabolites produced in vivo.
39,40
A mixture of hundreds of
13
C-isotope-labeled
metabolites, for example, can be produced within
a few hours by feeding microorganisms such as
yeast or bacteria with U-
13
C-labeled medium.
This approach is signi
cantly less expensive and
enables access to isotope-labeled standards for
the majority of metabolites of interest in biological
samples. As with commercially available SILIS
compounds, metabolite quantitation is performed
by mixing biological specimens with in vivo
isotope-labeled mixture and comparing mass
peak intensities of eachmetabolite with its labeled
counterpart or using the calibration plots
(
Figure 1
). Aone-time determinationofmetabolite
-
cantly lower matrix effects than those exhibited
in the biological samples. Without the presence
of labeled internal standards to offset the ion
suppression effect, one risks underreporting the
actual metabolic concentrations. In addition,
theMS signal can drift over time due to the
uctu-
ations in the ionization ef
ciency, and as a result
Search WWH ::
Custom Search