Biology Reference
In-Depth Information
extract the entire metabolome due to the broad
physiochemical nature of the metabolites.
Different polarity of the solvents bias the extrac-
tion ef
methanol solution containing the quenched cells
was used for extraction. Chemical extractions of
intracellular metabolites have included organic
solvents, alcohols, acids, and bases and hot and
cold temperatures. Detail procedures for PCA
and MeOH/CHCl
3
/H
2
O extraction of cells and
tissues are given by Beckonert et al.
25
and Salek
et al.
42
Extraction optimized for other analytical
methods
d
that is, GC-MS and HPLC-MS
d
may
not be suitable for NMR analysis because deriv-
atization in GC-MS and chromatography
columns may remove metabolites detectable by
NMR. Dietmair et al. tested 12 different cell
extraction methods using MeOH, ACN, 50:50
MeOH/CHCl
3
, EtOH, KOH, and PCA on
a recombinant Chinese hamster ovary cell line
(grown in suspension) and concluded that cold
extraction using 50% aqueous ACN was supe-
rior to other solvents, achieving over 95%
recovery for all chosen standard metabolites.
43
HPLC-MS was used to quantitate a standard
mixture of 29 metabolites that included nucleo-
tides and amino acids. The authors also
concluded that quenching with 60% methanol
resulted in intracellular metabolite leakage and
recommended quenching of fragile mammalian
cells with cold isotonic saline (0.9% w/v NaCl,
0.5
C). Using
1
H NMR, both Le Belle et al. and
Martineau et al. concluded that extraction with
MeOH/CHCl
3
/H
2
O was both more ef
ciency for different classes of metabolites
and interpretation of such results can lead to the
wrong biological conclusions.
38
The compromise
is an extraction technique that is ef
cient, repro-
ducible, and extracts the largest number of
metabolites possible so that the extract is repre-
sentative of the sample. Tiziani et al. compared
the precipitation of proteins in heat-inactivated
fetal bovine serum by the addition of perchlo-
ric acid (PCA), ACN, acetone, 50:50 MeOH/
CHCl
3
, and ultra
ltration removal with a 3 kDa
ltration was supe-
rior inmetabolite retention and reproducibility.
39
The same authors were able to recover the
nonpolar, protein-bound metabolite portion of
the serum that remained on the
filter and concluded that ultra
filter by washing
the
filter with methanol/water solution (3.2:1)
and extracting the wash solution with chloro-
form. Sheedy et al., working with both bovine
serum albumin spiked with a mixture of 15
metabolites and human serum, concluded that
the aqueous fraction from a 3:2 MeOD/CDCl
3
extraction resulted in higher level of metabolite
recovery compared to ultracentrifugation.
40
When using molecular weight
filters, glycerol
on the membrane, which serves as a preservative
and humectant, must
first be totally removed
with suf
cient multiple wash steps.
cient
and more reproducible than with PCA for
aqueous metabolites from adherent mammalian
cells.
44,45
The ratio of 2:2:1.8 MeOH/CHCl
3
/
H
2
O was worked out by Bligh and Dyer and
was originally developed for total lipid extrac-
tion.
46
Le Belle et al. used rat brain tissue and
rat astrocyte cells to compare MeOH/CHCl
3
/
H
2
O and PCA extraction and made their con-
clusions based on the yield of creatine, choline,
glutamine, glutamate, N-acetylaspartate, ala-
nine, and lactate. Perchloric acid, which is
also an oxidizing agent, was found to be
more ef
Cells and Tissue Extracts
Mammalian cell
cult
because of the more complex media that contain
a much higher concentration of the same endog-
enous metabolites found inside the cell and the
high metabolic turnover requiring fast quench-
ing of metabolic activities without damaging
the cells. Teng et al. introduced a fast quenching
technique where cells were quickly rinsed with
ice-cold phosphate-buffered saline (PBS, pH
7.4) and quenched with methanol.
41
The cells
were then detached using a cell lifter and the
extractions are dif
cient at inactivating enzymatic activi-
ties. Strong acids such as PCA can quickly
Search WWH ::
Custom Search