Simultaneous Extraction of Cellular Lipids and Water-
Soluble Metabolites: Evaluation by NMR Spectroscopy
Rakesh Kumar Tyagi, Anat Azrad, Hadassa Degani, Yoram Salomon
A method for simultaneous extraction of lipids and water-
soluble metabolitesfrom a single cell sample was developed
and optimized for NMR spectroscopy. Intermediary metabo-
lites in cultured M2R mouse melanoma cells and changes
therein in responseto challengewith melanotropin were stud-
ied by "P and I3C NMR. Cells were extracted with methanol,
chloroform, and water (i:l:l, v/v/v). The contents of the chlo-
roform and methanol-water phaseswere separated and quan-
titativety recovered. The contents of the upper and lower
phases compared well with the homologous fractions ob-
tained by perchloric acid and Folch's lipid extraction methods.
The pH of the extracts remained within the physiologicrange,
eliminating potential deleterious effect on cellular metabo-
lites. The water phase contained minimal amounts of salts,
making these extracts amenable to subsequent analytical
procedures. Obtaining lipid- and water-soluble metabolites
from the same sample enables characterization of metabolic
pathways that bridge the two cellular components in a quan-
titative manner.
Key words: perchloric acid extraction; lipid extraction; 31Pand
''C NMR spectroscopy; melanoma cells.
INTRODUCTION
The study of intact cultured cells by NMR spectroscopy
is more attractive because of the noninvasive nature of
the experimental approach and the fact that data are
acquired essentially under real time conditions (refs. 1,2,
and the Refs. therein). However, the limitations of this
method are (i) the inherently low sensitivity and conse-
quent low time resolution of the measurements; (ii) the
measurements, therefore, require relatively large masses
of cells that have to be superfused in the spectrometer
under sterile culture conditions; (iii) the information ac-
quired concerns almost exclusively water-soluble cell
metabolites. On the other hand, the work with cell ex-
tracts is an essential complementary part, supporting
observations made during metabolic studies of living
cells and tissues (for example, see ref. 3). Studies with
MRM 35.194-Mo (1996)
From the Departmentsof Hormone Research (R.K.T., A.A, Y.S.) and Chem-
ical Physics (H.D.), The Weizmann Institute of Science, Rehovot 76100,
Israel.
Address comespondence to: Yoram Salomon, Ph.D., The Department of
Hormone Research, The Weizmann lnstiute of Science, Rehovot 76100,
Israel.
Received June 6, 1995; revised August 18, 1995; accepted September 6.
1995.
This work was supported in part by research grants to H.D. and Y.S. from
the DKFZ-NCRD joint program in cancer research.
Yoram Salomon is the Charles and Tillie Lubin Professor of Hormone
Research.
Rakesh Kumar Tyagi is recipient of a postdoctoral fellowship from the
Feinberg Graduate School of the Weizmann Institute of Science.
0740-3194/96 $3.00
Copyright Q 1996 by Williams 8 Wilkins
All rights of reproduction in any form reserved.
194
cell extracts have several advantages. First, they do not
require tight coordination with scheduled cell cultures;
second, they inherently provide an improved resolution
of the spectrum because of the homogeneity of the sam-
ple solution; and third, the possibility of prolonging the
periods of data acquisition and the option to manipulate
sample concentrations enable the investigator to extend
the concentration range of the observed metabolites to
lower values. Despite the above advantages, great care
should be taken during extraction to minimize decompo-
sition of metastable metabolites.
The type of extraction method to be selected depends
greatly on the nature of the investigation and the analyt-
ical methods involved. In certain cases cell extraction is
the only method to study cellular metabolites (e.g., phos-
pholipids) that cannot otherwise be detected by NMR in
living cells. To analyze cellular lipids and/or their water-
soluble metabolites, it is necessary to use extraction pro-
cedures that quantitatively recover the metabolites of
interest. Among the available methods, perchloric acid
(PCA) is widely used for extraction of water-soluble cell
components, whereas the extraction of cellular lipids
uses organic solvents, preferentially chloroform and
methanol, as originally described by Folch et al. (4).
Methods for extraction of both the lipids and the meth-
anol-water-soluble metabolites are not in use, possibly
because of unsatisfactory recovery of these cellular com-
ponents. The need, therefore, of quantitative recovery of
both the lipid and water-soluble fractions from the same
biological samples by a single extraction method is obvi-
ous. This requirement arises in studies that deal with
enzymatic pathways that bridge both of these aspects of
the cell. These may involve intermediates of lipid and
phospholipid synthesis and/or their degradation prod-
ucts including those levels controlled by certain hor-
mone/neurotransmitter-regulated membrane signaling
systems (5-8).
In the present report we describe an extraction proce-
dure that fulfills these requirements and can be used for
simultaneous recovery of lipids and water-soluble com-
ponents from the same cell sample. The results were
evaluated by 31Pand 13C NMR. The present method is
simple and efficient and additionally provides the inves-
tigator with water-soluble fractions containing extremely
low levels of salts, a premium advantage for any subse-
quent analytical procedure.
MATERIALS AND METHODS
3-Isobutyl-1-methylxanthine (IBMX), [Nle4,~-Phe71a-
melanocyte-stimulating hormone ( [Nle4,~-Phe7]a-MSH),
and chloroform-d ('H-chloroform) were obtained from
Sigma Chemical Co. (St Louis, MO). Forskolin and
Chelex were purchased from Calbiochem (San Diego,
Lipid and Water-Soluble Extmcts
California) and BioRad Laboratories (Hercules, Califor-
nia), respectively. Deuterium oxide was a product from
E. Merck (Darmstadt, Federal Republic of Germany). Dul-
becco's modified Eagle's medium and culture medium
F12 were from Biological Industries (Beit Haemek, Isra-
el). Heat-inactivated horse serum was from Bio-Lab Ltd.
(Jerusalem,Israel). 13C-labeledcholine chloride (1,2-'3C),
ethanolamine-HC1 (l,2-l3C), and L-serine (3-13C) were
obtained from CIL (Woburn, Massachusetts). All other
chemicals used were of analytical grade.
M2R Cell Culture and Hormonal Stimulation
For studies of cell extracts, M2R mouse melanoma cells
were cultured as monolayers in Dulbecco's modified Ea-
gle's medium:F12 culture medium supplemented with
10% horse serum in a 8% CO, air atmosphere for 72 h
(9). On the day of the experiment, the cultures were
-90% confluent. For hormone stimulation studies, the
cell monolayers were incubated at 37°C for 30 min in
culture medium containing 0.1 mM IBMX, 1.0 pA4 fors-
kolin, and 0.1 f l [Nle4,~-Phe7]a-MSH. Corresponding
control cell cultures were treated with IBMX only. For
13C labeling experiments, M2R cells were cultured for 96
h in the same medium (free of choline and L-serine)in the
presence of 60 p M choline (1,2-13C),5 pM ethanolamine
(1,2-13C),and 0.25 mML-serine (3-13C).
Dual-Phase Extraction (DPE) Method
The cell culture plates (15 cm in diameter) were washed
twice with 10 ml of saline at room temperature. To each
plate, 4 ml of ice-cold methanol was added, and the
plates were kept covered on ice (5-10 min) to prevent
methanol evaporation during manipulation of the plates.
The cells were readily scraped with a rubber policeman
and pooled to their respective experimental group in
50-ml culture tubes. One volume of chloroform was
added, and the tubes were vigorously vortexed. One vol-
ume of water was then added: samples were vortexed
and left overnight at 4OC for phase separation. The final
ch1oroform:methanol:water ratio was 1:1:1 (v/v/v). The
upper methanol-water phase containing the water-solu-
ble cellular metabolites and the lower chloroform phase
containing the cellular lipids were carefully separated
and further treated as described below.
Recovery of the Water-Soluble Metabolites. To remove
the divalent ions, the methanol-water phase was treated
with Chelex (-10 mg/lO ml) and the beads removed by
centrifugation. The clear supernatant was lyophilized
and stored at -20°C until used. Lyophilization of the
methanol water extract required refreezing, once or
twice, because of the low freezing temperature of such
mixtures. For NMR analysis, the lyophilized sample was
dissolved in 0.5 ml of 'H,O containing 10 mM ethyl-
enediaminetetraacetate (EDTA) (pH 8.0),and the final pH
was adjusted with Tris to 8.2 in a microfuge test tube.
The tubes were then centrifuged, and the clear superna-
tant was transferred to the NMR test tube.
Recovery of Lipid Metabolites. The chloroform phase was
evaporated to dryness in a rotary evaporator. For N M R
analysis, the dried lipid sample was dissolved in 0.8 ml
of [2H]chloroformand 0.4 ml of 40 mkfmethanolic EDTA
195
(200 mM EDTA in water was adjusted to pH 6.0 with
CsOH and further diluted fivefold with absolute metha-
nol) and transferred to an NMR test tube (10). A typical
experimental group consisted of three culture plates.
PCA Extraction
Cell culture plates (15 cm in diameter) were washed
twice with 10 ml of saline at room temperature. To each
plate, 10 ml of ice-cold 5% PCA containing 1 mM Tris
was added. The plates were kept at 4°C for 30-60 min.
The PCA extract was collected and pooled in 50-ml cul-
ture tubes, leaving the cell monolayer intact. The extract
was neutralized to pH 7.6 with 5 N KOH. The tubes were
centrifuged at 2000 rpm for 10 min at 4OC to precipitate
the potassium perchlorate. The clear supernatant was
collected and treated with Chelex (-10 mg/lO ml extract)
to remove the divalent ions. After centrifugation (to re-
move chelex beads), the supernatant was lyophilized and
stored at -2OOC. For Nh4R analysis the lyophilized sam-
ple was dissolved in 1 ml of 'H,O containing 10 mM
EDTA (pH 8.0). Before NMR analysis, final pH was ad-
justed to 8.2 with Tris. A typical experimental group
consisted of three culture plates.
Lipid Extraction by Modified Folch's Method
The lipid extraction was performed according to modi-
fied Folch's method ( 4 , l l ) . The cell culture plates (15 cm
in diameter) were washed twice with 10 ml of saline at
room temperature. Then ice-cold methanol (4 ml) was
added to each plate and the plates kept (5-10 min) on ice
during the manipulation of the plates. The cells were
scraped with a rubber policeman and pooled to the re-
spective experimental group in 50-ml tubes. Two vol-
umes of chloroform were then added and mixed, fol-
lowed by 0.6 volumes of 0.1 M KC1. The tubes were
vigorously mixed and left overnight at 4OC for phase
separation. The lower chloroform phase containing the
lipids was carefully recovered and then evaporated to
dryness in a rotary evaporator. For NMR analysis, the
dried lipids were dissolved in 0.8 ml of [2Hlchloroform
and 0.4 ml of 40 mMmethanolic EDTA and transferred to
an NMR test tube. A typical experimental group con-
sisted of three culture plates.
NMR Spectroscopy
NMR experiments were performed with a Briiker AMX-
400 NMR spectrometer (Karlsruhe, Federal Republic of
Germany). "P spectra were recorded at 162 MHz. For
each spectrum, 150-2500 transients were accumulated
by either applying 90° or 4 5 O pulses, 20- or 2.4-s repeti-
tion time, respectively, and composite pulse proton de-
coupling during acquisition. 13Cspectra were recorded at
100.6 MHz, acquiring 400-1600 transients with 60"
pulses, 3.5-s repetition time, and composite pulse proton
decoupling during acquisition. The chemical shift of 13C
spectra of lipid extracts was referenced to methanol (at
natural abundance, 49.9 ppm). The extracts were ana-
lyzed at room temperature, and the assignment of the
signals was based on their chemical shifts. Glycerophos-
phocholine (0.48 ppm) or a-triphosphonucleotide
196 Tyagi et al.

(a-NTP) (-10.04 ppm) served as internal references for 0

n
calibrating the chemical shifts in 31P spectra of water-
soluble metabolites. The scale in 31P spectra of lipid
extracts were referenced to PtdCho at 0 ppm. 13C spectra
of water-soluble metabolites were referenced to p C1 of
glucose (at natural abundance, 96.8 ppm). The areas of
the signals were determined by the instrument integra-
tion mode.

RESULTS
In the DPE procedure, both the lipid and the water-
soluble cellular metabolites obtained from M2R cell cul-
tures were separated and quantitatively recovered. The
contents of the upper methanol-water phase of the DPE
method was compared with that obtained by the classical
perchloric acid extraction method, whereas the compo-
sition of the lower chloroform phase was compared with
that of Folch's lipid extraction method. The new DPE
procedure allowed us to recover the lipids and the water-
soluble metabolites from the same cell sample. Analysis
was performed by "P and '"C NMR spectroscopy.
A comparison of DPE with PCA extraction procedures
was done first using 3'P NMR spectroscopy. A typical "'P
NMR spectrum of M2R cellular phosphate metabolites
derived from the methanol-water phase of the DPE
method is shown in Fig. la. The main resonances were
assigned to phosphocholine (PC)and other phosphomono-
esters, phosphodiesters (PDE) (primarily glycerophos-
phoethanolamine and glycerophosphocholine), (a,p,y)-
triphosphonucleotides ((a,p,y)-NTP), a group of peaks
I
identified as uridine-diphosphatenucleotide-sugarderiv- 1

atives (UDPS)and Pi at about 3.0 ppm. Comparison of the PPm 1 0

DPE with PCA extraction procedures revealed that PDE
and UDPS, calculated as percent of total phosphates,
were higher (P < 0.01 and P c 0.02, respectively),
whereas NTP were significantly lower (P < 0.01) (Fig.
2a). We observed that after lyophilization, all water-sol-
uble phosphate metabolites except NTP could be solubi-
lized in absolute methanol. Therefore, the use of 50%
methanol (water-methanol phase) during DPE procedure
may explain the less efficient extraction of NTP. As evi-
dent from the standard deviation values of four different
extracts, the DPE method appeared to be relatively more
consistent in recovering the phosphate metabolites. After
lyophilization of the methanol-water phase, the dried
extract could be solubilized with much ease in smaller
volumes of the solvent, which was not the case with PCA
extracts in which variable amounts of insoluble salts
make solubilization difficult.
We have previously reported that, in the presence of
IBMX and a synergistic dose of forskolin, [Nle4,~-Phe7]
a-MSH induces a transient rise in the cellular concentra-
tion of 3',5'-cyclic adenosine monophosphate (CAMP),a
transient decrease in adenosine s'-triphosphate, and a
rise in the levels of three phosphomonoesters in M2R
mouse melanoma cells (12). The extracts prepared from
such hormonally stimulated cells by the two extraction
methods could be satisfactorily compared (Fig. 3). Be-
cause the extracts from DPE method could be solubilized
b
FIG. 1. Typical 31P NMR spectra of water-soluble phosphate and
phospholipid metabolites extracted from M2R mouse melanoma
cells by the DPE method. (a) For NMR analysis of the water-
soluble phosphate metabolites, the dried residue obtained from
three 15-cm culture plates was dissolved in 0.5 ml of 'H,O and
pH-adjusted to 8.2. In recording the spectrum, 3300 transients
were accumulated with 45" pulses and 2.4-s repetition time, using
exponential multiplication with a 3-Hz line broadening. Glycero-
phosphocholine served as the internal reference for calibrating the
chemical shifts. Abbreviations used are: PME, phosphomo-
noesters; PC phosphocholine; P,, inorganic phosphate; PDE,
phosphodiesters; GPE, glycerophosphoethanolamine; GPC, glyc-
erophosphocholine; (ct,p,y)-NTP, (a,p,y)-triphosphonucleotides;
UDPS, UDP-sugar derivative. (b) For NMR analysis of phospho-
lipids, the dried lipid residue was dissolved in 0.8 ml of [2H]chlo-
roform and 0.4 ml of methanolic EDTA. In recording the spectrum.
2200 transients were accumulated as in Fig. 1a, using exponential
multiplication with a 1-Hz line broadening. Phosphatidylcholine
served as the internal reference for calibrating the chemical shifts.
All other details were as described under Materials and Methods.
Abbreviations used are: PtdEtn, phosphatidylethanolamine;
plasm., plasmalogens; SPH, sphingomyelin; Ptdlns, phosphatidyl-
inositol; PtdCho, phosphatidylcholine; Ptds, phosphatidylserine.
in smaller volumes of 'H,O, it was possible to obtain
satisfactory NMR spectra in a relatively shorter times or
lower cell numbers.
Lipid and Water-Soluble Extracts 197

60 x
PCA extraction
8
c,
50 DPE
(P
c
0 40 ii
r
0

-a
Q
30 I e
c, T T -
0
+ 2c
r
0
8 10

0
QG

I I

i.I
V"

Folch's extraction
DPE

cn4
v)
0
.Ea3
n
c
f
CI

b
FIG. 2. Comparison of extraction efficiencies of water-soluble
phosphate metabolites by perchloric acid and DPE procedures (a)
and of phospholipids by the modified Folch's method and DPE
procedure (b) from M2R mouse melanoma cells. The spectra from
which the data were analyzed were recorded with 90"pulses and
20-srepetition time. The extract preparation and other experimen-
tal conditions used for quantification were as described under
Material and Methods and the legend to Fig. 1. The relative values
2 SD (n = 4) are presented against total phosphates. Abbrevia-
tions used are as in Fig. 1. PME' indicates phosphomonoesters
minus phosphocholine.
A comparison of DPE with Folch's lipid extraction pro-
cedure was done using 31PNMR spectroscopy. Figure 1b
shows a typical 31PNh4R spectrum of the phospholipids
derived &om the chloroform phase of the DPE method. The
main resonances from M2R mouse melanoma cell extracts
were assigned to cardiolipin, Phosphatidylethanolamine
plasmalogen, phosphatidylethanolamine, phosphatidyl-
FIG. 3. Typical 31PNMR spectra of [Nle4,0-Phe7]a-MSH-stimulat-
ed M2R melanoma cell extracts prepared by perchloric acid (a)
and DPE (b) methods. For NMR analysis, dried extracts from PCA
and DPE methods were dissolved in 1.0 and 0.5 ml of 'H'O,
respectively. In recording the spectra, 280 transients were accu-
mulated for the PCA extract while 140 transients for the DPE
extract with 90" pulses, 20-s repetition time, and 3-Hz line broad-
ening. Other details and abbreviations used are as in Fig. 1.
3',5'-cyclic adenosine monophosphate is indicated as CAMP.
serine, sphingomyelin, lysophosphatidylethanohnine,
phosphatidylinositol, phosphatidylcholine plasmalogen,
and phosphatidylcholine. Quantification of the results
showed that the phospholipids extracted by the DPE
method compared well with those extracted by modified
Folch's lipid extraction method [Fig. 2b). Similarly, '"C
Nh4R spectra of 13C-labeledlipid metabolites from the two
extracts revealed comparable results (results not shown).
198
An illuminating example for the application of the
present extraction procedure to quantitatively analyze
metabolic pathways was demonstrated by 'C labeling
experiments. Typical '"C and "'P spectra of the lipid and
water-soluble metabolites are shown in Fig. 4. The 31P-
13Csplitting because of the j-coupling of the phosphorus
and the nearest carbon-1 and the furthest carbon2 in the
31Pspectrum of the water phase (Fig. 4a) and lipid phase
(Fig, 4c) enabled us to determine simultaneously both the
total and '"C-labeled pools of PC (total pool was labeled
after 96 h) and of PtdCho (67% labeled after 96 h), re-
spectively. The 13C-''C splitting in the 13Cspectra dem-
onstrated the usefulness of I3C labeling in identifying
and quantitating the relative labeling of the metabolites
(PC, PE, PtdCho, PtdEtn). The simultaneous use of 3-13C-
serine and [ 1,2-'"C]ethanolamine enabled us to identify
the origin of the ethanolamine group in PtdEtn and study
the contribution from the base exchange pathway to Pt-
dEtn synthesis (Fig. 4d, insert) (13). The above detailed
information, which cannot be obtained by in vivo stud-
ies, is possible only with highly resolved spectra from
these cellular extracts.
DISCUSSION
The work presented here makes it evident that using
methanol, water, and chloroform at a ratio of 1:1:1 v/v/v
permits quantitative recoveries of both the lipid and the
water-soluble components from the same biological sam-
ple. A number of cellhissue extraction procedures and
31P
c line, [l ,2-13C]ethanolamine, and
ti
B
a rentheses indicate the position of
4 t l I the 13C label on choline and etha-
ppm 0 -10 -20 PPk i 0
a C
ppm 65 60 55 50 45
b
Tyagi et al.
their useful modifications have been applied for NMR
analyses but mostly reported in relation to studies of
either the water or the lipid phases. Although the Folch's
procedure (4) in its several variations (11, 14, 15) pro-
vides in principal both aspects of the cellular extracts, its
major reported applications relate to lipid extraction.
The contents of the water phase were probably found
unsuitable for further analysis because of unsatisfactory
recovery of cellular metabolites and the presence of ex-
cessive salts (mainly KCl) (4, 11)or HC1(11,14) added in
the course of the extraction protocol. No satisfactory
procedure that simultaneously recovers both the water-
soluble and water-insoluble components from the same
cellular source is presently in routine use.
Phospholipids, which constitute a major proportion of
cellular lipids are now known to play important func-
tional roles in intracellular signaling, secretion, mem-
brane transport, endocytosis, and cell fusion (5-8, 16-
18). In major cellular signaling mechanisms known to be
controlled by hormones, growth factors, or neurotrans-
mitters, including immunoresponsive processes in cells
of hematopoietic origin, a continuous flux of lipids and
lipid-derived metabolites crosses the solubility barrier.
Obviously many of these may be either water-soluble or
insoluble at the various physiologic scenarios studied.
Monitoring enzymatic pathways that bridge the two cel-
lular phases, therefore, requires simultaneous acquisi-
tion of both the lipid and the water-soluble metabolites
from the same cellular sample by a reliable and quanti-
tative method. The results obtained in our laboratory
FIG. 4. 31Pand 13C NMR spectra of
the water and lipid phases of M2R
melanoma cells cultured for 96 h
in t h e presence of [l,2-13C]cho-
[3-13C]serine.The numbers in pa-
nolamine moieties. Spectra a and b
are from the water phase, whereas
c and d are from the lipid phase of
t h e DPE procedure. (a)Assignment
of signals is as in Fig. la. From
insert PC:J 31p 13cm = 7.9 Hz and
J31p.l+r) = 4.4 Hz. (b) From insert
PC(1): Jl%,,l+j = 39.5 Hz and
JTlp-l+,) same as in (a). Unas-
signed peaks are from the extrac-
tion medium, e.g., trizma base,
EDTA, etc. (c) Assignment of sig-
nals is as in Fig. l b . From insert
PtdCho: = 7.0 HZ and
J31p-13c(,, = 4.8. (d) From insert
PtdCho(1): J13c(lj 13cm = 39.9 Hz
and J3lP_1%,) same as in (c).The
peak at 49.9 ppm is from the sol-
vent (methanol).From insert PtdEtn
(1): a = signal derived from
[3-13C]serineand b = signal de-
rived from 11,2-13C]ethanolamine
as reported previously (13).This re-
gion also includes plasm-RDEtn.
Lipid and Water-Soluble Extracts
using the DPE method seem reliable, consistent and com-
parable with those obtained with PCA and lipid extrac-
tion methods. We have demonstrated that with the new
extraction method it is possible to show hormone-in-
duced metabolic changes such as [Nle4,~-Phe7](r-MSH
stimulation of CAMP and phosphomonoesters in mela-
noma cells (12). Additionally, by using I3C labeling of
phospholipid precursors and recording 13Cand 31P spec-
tra of both the water and lipid phases, we were able to
study the pathways that lead to the synthesis of the
phospholipids. This approach can be further exploited to
measure simultaneously the kinetics of the manifold
pathways of phospholipid metabolism.
A major advantage of the new method is that the solid
residue derived from the entire water-soluble phase of
the cell extracts is extremely small and can, therefore, be
taken up in a concentrated form in a small volume for
rapid NMR analysis. Moreover owing to the low salt
contents, larger volumes of these extracts could be fur-
ther fractionated and analyzed by ion-exchange thin
layer chromatography and by high-performance liquid
chromatography. The PCA extraction method may in-
duce the hydrolysis of certain acid-labile cell constitu-
ents such as NADPH, NADH, inositol phosphates, fruc-
tose 2,6-diphosphate, or PCr (3, 19,20).To minimize this
uncontrollable deficiency, PCA extraction is best per-
formed in the cold, whereas in the DPE procedure the
acidification of the extract is entirely eliminated and the
pH of the extracts remains always near the physiologic
range. In the PCA extraction method, a huge precipitate
of potassium perchlorate is formed when the initial ex-
tract is neutralized with KOH. On further cooling of the
supernatant and with each additional freezing-thawing
step, as well as in its final lyophilized form, there is
always continued precipitation of potassium perchlorate,
resulting in undesirable alterations of extract contents
and volume. Furthermore, in small or dilute samples, the
loss of relevant material may be significant. This fact may
explain the larger variance in the sample composition of
the PCA extraction method compared with the DPE
method (Fig. 2a). Metabolite composition (in percent of
total phosphates) as observed by the two methods
showed a different distribution (Fig. 2a). However, it
should be noted that the individual values expressed as
percent are interdependent and add up to 100%. Conse-
quently the less efficient extraction of PDE and UDPS by
the PCA method resulted in an apparent increase in
%NTPs. Indeed, in experiments in which the extraction
efficiency of PDE and UDPs by the two methods was
more similar, NTP levels were closer. Another example
of a deficiency of the PCA extraction procedure was
noted by Pianet et al. @I),who observed larger increases
in phosphodiester resonances in 31PNMR spectra when
studying living C6 glioma cells but not in spectra of their
respective PCA extracts. These investigators speculated
that hydrolysis of these phosphodiesters during the ex-
traction process may explain this discrepancy. Further-
more, not all perchlorate is precipitable (22). In addition
the PCA extracts invariably retain endogenous salts as
well as residual NaCl used in the washing procedure.
These interfere heavily with subsequent analytical pro-
cedures. To overcome this type of problem, several in-
199
vestigators have attempted to desalt biological extracts,
but the strategies suggested worked only with partial
success or required special apparatus (19, 23, 24). The
simple strategy of salt exclusion used in the DPE method
proved to be simpler and more effective compared with
suggestions made in earlier reports. Thus, the serious
interference caused by the presence of salts during the
chromatographic procedures and other analytical proce-
dures could be eliminated.
The classical Folch’s lipid extraction method has been
used with several modifications for lipid extraction from
cells and tissues (11, 14). The use of salts during the
extraction procedure was justified by the suggestion that
their inclusion improved not only the efficiency of ex-
traction of some acidic lipids but also the washing steps
involved (4). However, the quantitative results as pre-
sented here by NMR with respect to the phospholipids
derived from the two extraction methods appeared to be
independent of exogenous salts (Fig. 2b). Thus, within
the limits of experimental deviations, the relative levels
of phospholipids derived from the chloroform phase of
the DPE method were comparable with the ones obtained
by the modified Folch’s lipid extraction method. Al-
though we have applied the present protocol mainly to
the cells in cultures, it should also be easily adaptable to
the equivalent tissue samples.
ACKNOWLEDGMENTS
The authors thank Mircea Greenberg for help with NMR spec-
troscopy and Rachel Benjamin for excellent secretarial assis-
tance.
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