Biology Reference
In-Depth Information
(A)
Dilution
Cell removal:
centrifugation or
filtration
Bacterial growth
inhibition
Urease treatment
to remove urea
Anticoagulant
selection
Protein removal
Solvent extraction
Evaporation/
reconstitution
Rinsing
Quenching
Homogenization
Solvent extraction
Evaporation/
reconstitution
Protein removal
Solvent extraction
Evaporation/
reconstitution
Serum or
CSF
Urine
Plasma
Tissue
(B)
GC-
MS
LC-
MS
Particulate removal
Control pH and ionic
strength
Addition of
deuterated solvent
Reduce injection
solvent strength
Minimize ion
suppression
Protein removal
Water removal
Derivatization
Protein removal
NMR
FIGURE 2
Summary of critical sample preparation steps that are typically included in metabolomic analysis of biological
fluids and tissues depending on (A) sample type (B) instrumental analysis method. Parameters shown in black text are
mandatory, and the inclusion of parameters shown in blue text is optional.
When a quenching step is omitted, residual
enzymatic activity can signi
insight in residual enzymatic activity of
samples.
23
Inadequate metabolism quenching,
employed in most common strategies for blood
and urine samples, puts a great emphasis on
sample storage at low temperatures, preferably
at
e
80
C immediately after collection, but this
may not ensure accurate levels of all metabolites
and the results for labile metabolites should be
interpreted with extreme caution.
24
In summary, the rationale that metabolism
quenching is not important for the majority of
bio
cantly change
metabolite levels prior to analysis, causing poor
correlation between the observed metabolome
and the true metabolome at the time of sampling.
For instance, residual enzymatic activity in refrig-
erated rat plasma resulted in increased levels of
choline, glycerol, tyrosine, and phenylalanine.
19
For
cerebrospinal
fluid,
rapid centrifugation
immediately (
5 min) after collection to remove
white blood cells and snap-freezing (
<
2hours)
were important to preserve accurate metabolite
levels.
20,21
After delayed storage of 30 and 120
min, numerous metabolites (24 and 46, respec-
tively, out of a total of 57 known metabolites
detected) showed signi
<
uid studies may be acceptable when dealing
with very stable metabolites, but such an
approach is extremely questionable for labile
metabolites prone to degradation or conversion.
Although unstable and labile metabolites may
constitute only a small portion of the metabo-
lome, they may represent important biomarkers
or may complicate the search for true biomarkers
if differences in these metabolites arise due to
inadequate sample preparation. For example,
glutathione, adenosine, and inosine have all
been reported as possible biomarkers of cancer
25
cantly elevated levels
due to unquenched enzymatic activity. The moni-
toring of the residual enzymatic activity is highly
recommended even in methanolic solutions, as
certain enzymes such as hydrolases and phospha-
tases may remain active.
22
For urine, monitoring
of substrates for glutamyltransferase, alkaline
phosphatase, and urease can provide useful
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