Characterization of
1
H NMR detectable mobile lipids
in cells from human adenocarcinomas
Anna Maria Luciani
1
, Sveva Grande
1
, Alessandra Palma
1
, Antonella Rosi
1
, Claudio Giovannini
2
,
Orazio Sapora
3
, Vincenza Viti
1
and Laura Guidoni
1
1 Dipartimento di Tecnologie e Salute and INFN Gruppo Collegato Sanita
`
, Istituto Superiore di Sanita
`
, Rome, Italy
2 Dipartimento di Sanita
`
Pubblica Veterinaria e Sicurezza Alimentare, Istituto Superiore di Sanita
`
, Rome, Italy

1
H NMR ML
signals of cultured tumour cells, specifically HeLa and
Keywords
cell cycle; cell metabolism; lipids; magnetic
resonance spectroscopy; tumour cell lines
Correspondence
L. Guidoni, Dipartimento di Tecnologie e
Salute, Istituto Superiore di Sanita
`
, 00161
Rome, Italy
Fax: +39 06 4938 7075
Tel: +39 06 4990 2804
E-mail: [email protected]
(Received 5 November 2008, revised 15
December 2008, accepted 22 December
2008)
doi:10.1111/j.1742-4658.2009.06869.x
Magnetic resonance spectroscopy studies are often carried out to provide
metabolic information on tumour cell metabolism, aiming for increased
knowledge for use in anti-cancer treatments. Accordingly, the presence of
intense lipid signals in tumour cells has been the subject of many studies
aiming to obtain further insight on the reaction of cancer cells to external
agents that eventually cause cell death. The present study explored the rela-
tionship between changes in neutral lipid signals during cell growth and
after irradiation with gamma rays to provide arrest in cell cycle and cell
death. Two cell lines from human tumours were used that were differently
prone to apoptosis following irradiation. A sub-G1 peak was present only
in the radiosensitive HeLa cells. Different patterns of neutral lipids changes

pattern. Effects on cell cycle frequency were also
observed. ML signal intensity modulation was tenta-
tively related to modulation of lipid metabolism.
Results
Cell spectra were first examined for signal quantifica-
tion after spectral assignment. Subsequently, changes
in signal modulation were monitored when cell growth
was arrested by means of treatment with ionizing radi-
ation. Finally, a model to fit signal intensity modula-
tion was proposed.
Analysis of
1
H NMR spectra – spectral
assignments and quantification
Both cell lines displayed very similar spectral features.
Besides other signals, the characteristic peaks from
MLs were observed to be in agreement with the data
available in the literature for cancer cells [1–4]. Under
the present experimental conditions, the intense ML
signals can be attributed to the fatty acid chains of
neutral lipids, mostly TGs, in agreement with the data
available in the literature [1,15] and on the basis of
our previous observations showing that, in these cells,
TG peaks are more intense in the lipid extract spectra
derived from cells with high ML signals compared to
spectra with low ML signals [13]. This point will be
discussed further below.
Figure 1 shows an example of the
1
H NMR spectra

able in the literature [15] and previous observations
[13]. The cross peak at 4.07–4.24 p.p.m., generated by
protons in the glycerol backbone of TG, was also visi-
ble only in ML rich spectra (T). Details with respect to
this signal are provided in Fig. 1B (insert), which
shows the characteristic cross peak from the geminal
protons of carbons 1 and 3 of glycerol in TG [1].
Cross peaks of lipids, including the T signal, were
absent in cells characterized by low ML signals in 1D
spectra (Fig. 1B¢). Very similar behaviour was
observed in HeLa cells according to previously
reported data [4,13].
Signal assignments in cell spectra were performed
after comparison with the spectra from lipid and per-
chloric acid (PCA) extracts, derived from cell samples
grown and harvested under similar conditions, and
with compound spectra. Assignments from the litera-
ture were also taken into account [1–4,16]. Figure 2
shows typical spectra (1D and 2D COSY) from lipid
and PCA extracts both relative to a MCF-7 cell
sample with high MLs. It is worth noting that, in the
2D COSY spectra from extracted lipids, the glycerol
geminal cross peaks of 1,3-glycerol protons of TG and
of 1-glycerol protons for phospholipids (PL) are clearly
separated, in agreement with the data available in the
literature [1].
For peak assignments and intensity quantification,
deconvolution of 1D spectra and integration of 2D
cross peaks was performed as described in the Experi-
mental procedures. The signal intensity refers to the

A. M. Luciani et al.
1
H NMR of mobile lipids in tumour cells
FEBS Journal 276 (2009) 1333–1346 ª 2009 The Authors Journal compilation ª 2009 FEBS 1335
For 1D spectra, deconvolution of spectra of the type
shown in Fig. 1A¢, with ML signals of a very low
intensity, was performed as a first step. Deconvolution
of spectra of the type shown in Fig. 1A, with intense
ML signals, was then performed starting from the lines
and parameters previously found, by adding the signals
from MLs. A typical deconvolution pattern with the
used resonances is shown in Fig. 3A. The correspond-
ing parameters are given in Table 1. Experiments con-
ducted on different samples derived from the same
culture (at least three samples) demonstrated that the
SD from this procedure did not exceed 0.01 p.p.m. for
chemical shifts and 10% for linewidths and intensity
ratios (IRs). On the other hand, the variability of
signal intensities, especially for MLs, exceeded the
measurement error in spectra from samples derived
from different culture, even when cells were harvested
under similar growth conditions. For this reason, the
spectral behaviour over time was compared among
samples obtained by cells harvested at different days
from the same seeding.
Cholesterol peaks were present in lipid extracts at
0.70 and 1.03 p.p.m. in the 1D spectra (Fig. 2A) and
with the typical cross peak at 0.87–1.50 p.p.m. in the
2D spectra (Fig. 2B). In spectra from cell samples, we
could therefore assign the peak at 0.71 p.p.m. (Fig. 3A

as those presented in Fig. 3. Values were obtained from the spec-
tra of three different samples derived from the same culture. The
standard deviation was 0.01 p.p.m. for chemical shift values and
10% for linewidths and IR. Chemical shifts are referring to lactate
methyl; IR are calculated with respect to M2 in 1D and to
Lys1 + Ala in the 2D spectra.
1D d (p.p.m.) Dm (Hz) IR
Chol 0.71 40 0.15
ML1 + M1 + Chol 0.89 24 0.16
M2 0.95 28 1.00
Chol 1.03 20 0.05
M3 1.22 46 0.14
ML2 1.28 17 3.26
ML3 1.31 23 2.70
LAC 1 1.32 8.3 0.58
LAC 2 1.34 7.1 0.67
M5 1.40 36 1.30
M6 + Chol 1.48 22 0.13
ML4 1.58 38 0.89
M7 1.70 39 0.55
Broad 2.20 870 3.10
2D d (p.p.m.) IR
Lys1 1.70–3.00 1.00
Lys2 1.46–1.67 0.28
A 0.89–1.27 4.50
E 1.31–1.55 2.20
B 1.34–2.00 2.70
F 1.58–2.22 2.47
Lino 0.96–2.03 0.72
Chol 0.87–1.51 0.24

of large molecules because they are characterized by
large linewidths (Table 1 and Figs 2 and 3). Work is in
progress to clarify the nature of these structures.
Signal intensities were also measured in the 2D spec-
tra. A typical 2D COSY spectrum is shown in Fig. 3b,
including the details of the cross peaks examined. The
peaks chosen for evaluation are framed with rectangles
that denote the areas used for volume integration.
When very intense ML signals are present in the spec-
tra, the signals related to unsaturated fatty acids also
are evident, and are more intense in MCF-7 than in
HeLa cell samples. Besides the cross peaks resulting
from the connectivity of the vinyl protons (at
5.35 p.p.m.) to the allylic protons (at 2.05 p.p.m.) in
monounsaturated chains and to the bis-allylic protons
(at 2.80 p.p.m.) in polyunsaturated fatty acids (not
shown), the cross peaks at 1.64–2.09 p.p.m. and 1.68–
2.24 p.p.m., attributed to arachidonic acid chains, and
at 0.93–2.04 p.p.m., attributed to linolenic acid chains
on the basis of a comparison with lipid extracts and
from the data available in the literature, are also
clearly visible in Fig. 1B,B¢.
Experiments on different samples derived from the
same culture (at least three samples) were also exam-
ined to assess measurement errors in the 2D cross peak
integration. Under these conditions, errors on cross
peak volumes did not exceed 10%, whereas the vari-
ability of integral values exceeded this error in the
spectra from samples derived from different cultures,
even when cells were harvested under similar growth

with different characteristics in the two cell lines,
according to previous observations [14]. Figure 4
shows the cell counts as a function of time for one
representative experiment in HeLa and MCF-7 cells.
Compared to control samples, the differences in cell
counts were larger in HeLa than in MCF-7 cells.
Similar behaviour was observed in at least three
independent experiments.
To assess cellular transcriptional responses to radia-
tion-induced DNA damage, we examined cell cycle
arrest in MCF-7 and HeLa cells at 1, 2 and 3 days
after treatment. Both cell lines underwent cycle arrest
upon irradiation, with different characteristics.
Figure 4A¢,B¢ shows the percentage of cell phases of
both cell lines observed after 2 days after irradiation at
20 Gy. Although both cells were blocked in G2 ⁄ M,
HeLa cells displayed a remarkable decrease in the
A. M. Luciani et al.
1
H NMR of mobile lipids in tumour cells
FEBS Journal 276 (2009) 1333–1346 ª 2009 The Authors Journal compilation ª 2009 FEBS 1337
G1 phase, whereas MCF-7 cells showed G1 block and
a decreased percentage in the S phase compared to
control samples. Irradiated HeLa cells showed an
intense sub-G1 peak (> 20%), indicating DNA frag-
mentation and the occurrence of significant apoptosis.
This observation is in agreement with previous data
obtained in the same cells by monitoring apoptosis as
the externalization of phosphatidyserine [14].
1

shows these differences for samples examined 2 days
after irradiation.
Figure 6 shows the glycerol region from two repre-
sentative 1D spectra of total lipids extracted from
control and irradiated HeLa (Fig. 6A,A¢) and MCF-7
(Fig. 6B,B¢) cells. A significant change in relative inten-
sity of TGs (glycerol proton centred at 4.32 p.p.m.)
versus PLs (glycerol proton centred at 4.42 p.p.m.)
was found in irradiated samples compared to controls.
Particularly, TG signals were depressed in HeLa cells
(Fig. 6A,A¢), whereas an increase was evident in
MCF-7 cells (Fig. 6B,B¢). Deconvolution of 1D spectra
was performed to provide the relative intensities of TG
versus PL, which were calculated on sn-1 and sn-3
glycerol signals centred at 4.32 p.p.m. for TG and sn-1
glycerol signals at 4.42 p.p.m. for PL (Fig. 6C). In this
experiment, the calculated relative concentration
of TG versus TG + PL was 15% and 12% in MCF-7
A
A′
B
B′
Fig. 4. Number of HeLa (A) and MCF-7 (B) cells (N) as a function of time after irradiation for both control (h) and irradiated ( ) samples.
The solid black line is the fit with an exponential function. Percentage of MCF-7 (A¢) and HeLa (B¢) cells in the different cell cycle phases,
measured 2 days after irradiation. One representative experiment is reported for both control and irradiated samples.
1
H NMR of mobile lipids in tumour cells A. M. Luciani et al.
1338 FEBS Journal 276 (2009) 1333–1346 ª 2009 The Authors Journal compilation ª 2009 FEBS
and HeLa cells, becoming 20% and 8%, respectively,
after irradiation. The standard deviation in repeated

In particular, more relevant changes in GPC ⁄ PC ratios
were observed in irradiated MCF-7 cells (Fig. 7B,B¢)
with respect to HeLa cells (Fig. 7A,A¢), indicating the
different equilibrium of catabolism versus anabolism.
Table 3 reports on the intensity of changes of the
choline-based metabolites for the two cell lines after
irradiation resulting from fittings of at least three
different spectra.
Time (day)
Time (day)
Time (day)
Time (day)
A/(Lys + Ala) c
A/(Lys + Ala) i
A
B
A′
B′
C
Fig. 5. Intensity modulation of ML signals
from a representative experiment (1D and
2D
1
H NMR data) for HeLa (A, A¢) and
MCF-7 cells (B, B¢). Spectra were acquired
at different days from seeding for both
control (h) and irradiated (
) samples
(D = 20 Gy). Errors obtained from spectral
fitting (1D) and integration (2D COSY) are

production over time must be therefore envisaged.
There is a growing body of evidence indicating that
lipid metabolism possesses an articulated role with
respect to maintaining cell equilibrium, which takes
into consideration both PL synthesis ⁄ breakdown and
TG metabolism [18–20]. We may infer that there are
two mechanisms inside the cell: one relative to the pro-
duction of ML (and TG) with a rate constant R
p
and
one relative to the consumption of ML (and TG) with
a rate constant R
c
. The signal that we observe in the
NMR spectra is due to the net accumulation of reserve
lipids and its rate, dML ⁄ dt, is given by the difference
of the rate of lipid production R
p
and the rate of lipid
consumption R
c
:
dML=dt ¼R
p
ÀR
c
ð1Þ
We may assume that both rates R
p
and R

time.
By integrating Eqn (1), we obtain a second degree
polynomial function for the lipid accumulation:
MLðtÞ¼m
1
t
2
þm
2
tþm
3
ð4Þ
where m
1
=(c
2
) p
2
) ⁄ 2, m
2
=(p
1
) c
1
) and therefore
are related to the equilibrium between lipid production
and consumption, and m
3
is the starting ML value.
The best fit with Eqn (4) of data ML ⁄ M and

1
H NMR
spectrum for sn-2 glycerol protons of TGs (couple of doublets
centered at 4.32 p.p.m., coupling 12 Hz and 4 Hz) and PLs (couple
of doublets centered at 4.42 p.p.m., coupling 12 and 3 Hz). Accord-
ing to this deconvolution pattern, in this spectrum, the ratio
TG ⁄ (PL + TG) was 0.34.
1
H NMR of mobile lipids in tumour cells A. M. Luciani et al.
1340 FEBS Journal 276 (2009) 1333–1346 ª 2009 The Authors Journal compilation ª 2009 FEBS
The minimum value t
m
of the parabolic fitting curve,
representing the time value for which production and
consumption rates are equal, and the corresponding
lipid value ML
m
are also reported.
1D and 2D data were in good agreement, although
the starting intensity values and values at the minimum
were different, reflecting different pools of ML inside
the cells. By changing the seeding density, the minimum
of the curve was shifted, but the shape of the curves
did not change. It is worth noting that m
1
was always
positive (i.e. c
2
was always greater than p
2

reference signal (C¢).
Table 2. Mean values of parameters d (p.p.m.), Dm (Hz) and IR
after deconvolution of 1D spectra from PCA extracts of HeLa cells
(Fig. 7C,C¢). Values were obtained from the spectra of three differ-
ent samples derived from the same culture. The standard deviation
was 0.005 p.p.m. for chemical shift values and 10% for linewidths
and IR.
D (p.p.m.) Dm (Hz) IR
M (reference) 0.939 25.0 1.00
Choline 3.219 1.88 0.45
PC 3.226 2.20 0.70
GPC 3.236 1.65 0.53
Table 3. Mean values (three independent experiments) of IRs of
the choline-related metabolites from PCA extracts of HeLa and
MCF-7 cells for control and irradiated samples. The standard devia-
tion was 10%.
GPC PC Cho
HeLacells
Controlsample 0.43 0.71 0.50
Irradiated sample 0.28 0.86 0.72
MCF-7 cells
Control sample 1.12 1.31 0.60
Irradiated sample 0.90 0.77 0.63
A. M. Luciani et al.
1
H NMR of mobile lipids in tumour cells
FEBS Journal 276 (2009) 1333–1346 ª 2009 The Authors Journal compilation ª 2009 FEBS 1341
Although the t-test showed that differences were not
significant between the two groups (HeLa and MCF-
7), m

great increase of ML signals with respect to controls
(Fig. 5B,B¢). In some experiments conducted on
MCF-7 cells, the minimum of the parabolic curve at
time > 0 was no more evident and m
2
became posi-
tive. Finally, t
m
shifted to lower values in MCF-7 cells
and the ML
m
value was considerably higher in
irradiated MCF-7 cells compared to controls.
Discussion
The appearance of intense signals from bulk methylene
of fatty acid chains in high resolution
1
H NMR spec-
tra of cells has been studied subsequent to the first
observations being made in cancer cells, lymphocytes
and developing cells. On the other hand, a correlation
of the intensity of these signals with metabolic parame-
ters is not straightforward. Some studies noted that
the signal intensity of bulk methylene is influenced by
cell proliferation, as T lymphocyte activation [21] and
in tumour cells by the different proliferation state
[4,22]; other studies found that the onset of apoptosis
correlates with the increase of lipid signals, whereas
others did not [5–9]. Finally, some studies found that
these signals can be affected by extreme pH conditions,

lipid values (ML
m
) are also reported. Bars
represent the mean ± SD of ten indepen-
dent experiments for each cell line.
5
0
–5
–
10
5
0
–5
5
0
–5
5
0
–5
–
10
Fig. 9. Parameters m
1
, m
2
and m
3
obtained
from fitting with Eqn (4) ML data, as in
Fig. 5, for irradiated (I) and non-irradiated (C)

the S and G2 phases while trying to repair the dam-
aged DNA, with a subsequent block in the G2 phase,
at the expense of G1 (Fig. 4A¢ ). On the other hand,
HeLa cells undergo apoptosis after irradiation, accord-
ing to that previously reported for other cells lacking
p53 [23].
MCF-7 cells were found to be more resistant to irra-
diation (Fig. 4B), displaying an absent or a very low
apoptotic percentage compared to controls [14,24],
despite the presence of p53 and the subsequent block
in G1 (Fig. 4B¢). Cells are also arrested in G2, whereas
the S phase is markedly reduced (Fig. 4B¢). Cells are
unable to enter mitosis, although they escape an apop-
totic fate. This finding is in agreement with other stud-
ies that also found very low apoptosis and arrest in
G2 and G1 in these cells after irradiation, whereas the
same cells could undergo apoptotic death upon treat-
ment with daunorubicin [24] or buthionine sulphoxi-
mine [14,24].
The two cell lines showed similar behaviour with
respect to the intensity of variation in bulk methylene
signals (ML) when kept in culture, displaying high
intensities, followed by a decrease and subsequent
recovery. This was not true after proliferation arrest
induced by irradiation because MCF-7 cells displayed
a faster recovery of ML signals with respect to non-
irradiated cells, whereas the opposite was the case for
HeLa cells (Fig. 5).
On comparison, the ML intensity in cells (Fig. 5)
correlated with TG intensity in extracted lipids both

and c
1
> p
1
. This means that the consump-
tion lipid rate starts faster than production, but slows
down over time more than production. This latter
effect produces the very intense ML ⁄ TG signals that
are observed in the final days in culture. On the other
hand, the high rate of proliferation in the exponential
growth curve is accompanied by a high consumption
rate of TG (i.e. the ML ⁄ TG signal in cells decreases
shortly after seeding) (Fig. 5). This can be explained
by an increased fatty acid demand for PL synthesis as
cells proliferate and is in agreement with the high PC
signals observed in PCA extracts for both cell lines
shortly after cell seeding (not shown). It is worth not-
ing that the reduction in TG, hydrolyzed either due to
energy demands or as a source of fatty acids, occurs
when cells are in the presence of fresh growth medium,
from which both energy and fatty acids can be taken.
When cell growth slows down, the PL demand also
decreases, allowing the storage of fatty acids in TG. It
has been demonstrated that TG may be accumulated
through the diversion of PL synthesis from diacylglyce-
rols when phosphatidylcholine synthesis is reduced or
in the presence of phosphatidylcholine degradation
[18,25]. In particular, inhibition of the final step of
phosphatidylcholine synthesis via the Kennedy path-
way may divert diacylglycerol molecules into TG.

we may infer that c
1
tends to become lower than p
1
after growth arrest, at least in this cell line. The effect
was less intense in HeLa cells, probably due to the ten-
dency of the S phase to remain high in this cell line, in
contrast to MCF-7 cells (Fig. 4A¢,B¢) where the
S phase is low after irradiation. Due to a net accumu-
lation of PL synthesis in the S phase, if a reduced
S phase is paralleled by a reduction of the final step of
PL, TG accumulates, as observed in MCF-7 cells after
irradiation. A low PL synthesis in MCF-7 cells is also
sustained, as demonstrated by the observation in PCA
extracts (Fig. 7) where growth-arrested MCF-7 cells
show low PC and increased GPC, indicating an imbal-
ance between PL synthesis and degradation and, con-
sequently, an increase of TG. Other studies suggest
that the simultaneous accumulation of ML and GPC
may be linked to PL catabolism [7].
Modulation of ML intensity can then be ascribed to
an equilibrium between the build up and consumption
of these molecules due to the cellular state. In control
cells, these changes can only be ascribed to a depletion
of TG when cells are actively duplicating and a subse-
quent restoration of TG storage when cells approach
confluence. The timing of this equilibrium is influenced
by cell seeding conditions. On the other hand, when
cells stop growth due to the effects of irradiation, dif-
ferent trends are offered by the two cell lines.

and consumption that depends on the proliferative
state of the cell.
Experimental procedures
HeLa cells, kindly provided by G. Aquilina (Istituto Superi-
ore di Sanita
`
, Rome, Italy) and purchased from the ICRF
Cell Production (Clare Hall, UK) and MCF-7 cells, kindly
donated by S. Meschini (Istituto Superiore di Sanita
`
,
Rome, Italy) and purchased from ATCC (Manassas, VA,
USA) were grown as described previously [14]. Cells of
both cell lines were routinely seeded in 175 cm
2
flasks at a
density of 4 · 10
5
cells per flask in 50 mL of medium.
Cell culture flasks were irradiated with 20 Gy
60
Co
gamma rays. Cells were detached at different times after
irradiation (day zero = 2 h after irradiation), counted and
samples were prepared for NMR measurements, as
described below.
Cell cycle measurements
Cells were detached, washed and suspended in NaCl ⁄ P
i
at

with 30% (weight : volume) cold PCA. After 30 min, the
1
H NMR of mobile lipids in tumour cells A. M. Luciani et al.
1344 FEBS Journal 276 (2009) 1333–1346 ª 2009 The Authors Journal compilation ª 2009 FEBS
solution was neutralized with KOH and centrifuged. The
liquid phase was then lyophilized.
Before NMR measurements, samples were suspended in
D
2
O and the pH was adjusted to 7.4. Lipid extracts were
prepared by suspending the pellet obtained from PCA
extracts preparation in distilled water. The solution was
then lyophilized and the lyophilized powder dissolved in a
2 : 1 CDCl
3
⁄ CD
3
OD solution for NMR measurements, as
described previously [13]. All reagents for cells were pur-
chased from Sigma (St Louis, MO, USA) and deuterated
NMR reagents were purchased from Cambridge Isotope
Laboratories, Inc. (Andover, MA, USA).
1
H NMR measurements
To perform NMR measurements on cells, a pellet of
approximately 5 · 10
6
cells was suspended in NaCl ⁄ P
i
with

fixed and applied to all the spectra. Mean ± SD values
calculated from 1D deconvolution and 2D integration of
at least five spectra of samples prepared from the same cell
culture were used to assess the error on the single mea-
surement. Fitting of data to obtain model parameters was
performed using origin software (OriginLab Corp.,
Northampton, MA, USA).
Statistical analysis
Student’s t-test was applied to the two-sample groups to
compare variations in intensity of the control and irradi-
ated samples. Student’s t-test works under the assumption
of a Gaussian distribution of data, but also works remark-
ably well for distributions that are not accurately Gaussian
[27]. Variations in intensity of the examined signals are not
linear with time and ⁄ or cell proliferation [13]. It is therefore
necessary to examine the signal intensity for samples of
cells seeded and harvested under similar conditions. Fur-
thermore, we always irradiated cells under similar growth
conditions. Under this experimental set-up, the value distri-
bution appears to be Gaussian, although the limited num-
ber of experiments (approximately ten for each time
interval) cannot guarantee the presence of a perfect Gauss-
ian shape. The second hypothesis necessary to perform
Student’s t-test requires that the variance of the two samples
is equal. By estimating the variance from the standard devia-
tions and by using the F distribution [27], we could not find
any statistical difference in the variance when examining
control and irradiated samples. This indicates that the use of
Student’s t-test is compatible with the examined data.
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