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Chemistry and Physics of Lipids 150 (2007) 229–238

Apoptosis-associated changes in the glycerophospholipid

composition of hematopoietic progenitor cells
monitored by 31P NMR spectroscopy and
MALDI-TOF mass spectrometry
Beate Fuchs a,∗ , Jürgen Schiller a , Michael A. Cross b
aUniversity of Leipzig, Medical Faculty, Institute of Medical Physics and Biophysics,
Härtelstr. 16-18, D-04107 Leipzig, Germany
b Department of Hematology/Oncology & Interdisciplinary Center of Clinical Research,
Inselstr. 22, D-04103 Leipzig, Germany
Received 21 March 2007; received in revised form 27 August 2007; accepted 28 August 2007
Available online 1 September 2007

Abstract
Apoptosis, or programmed cell death, plays an important role in development and in tissue homeostasis and is assumed to
be accompanied by changes in the composition of cellular glycerophospholipids (GPL). We have applied a combination of 31 P
nuclear magnetic resonance spectroscopy and matrix-assisted laser desorption and ionization time-of-flight (MALDI-TOF) mass
spectrometry (MS) to the analysis of organic extracts of hematopoietic progenitor cells undergoing the physiologically relevant
process of apoptosis following growth factor withdrawal. The combined application of these methods enables the quantitative
analysis of all glycerophospholipid classes and reveals changes in the acyl chain compositions from crude cell extracts. Using
these techniques, an increase in the ratio of ether-linked glycerophospholipids to diacyl-glycerophospholipids during apoptosis was
observed. The relative decrease in the membrane diacyl-phosphatidylcholine (PC) levels was found to correlate with increased
concentrations of the corresponding lysophosphatidylcholine (LPC) present in the medium.
© 2007 Elsevier Ireland Ltd. All rights reserved.

Keywords: 31 P NMR; MALDI-TOF MS; FDCPmix cells; Apoptosis; Diacyl-glycerophospholipids; Ether-linked glycerophospholipids

1. Introduction the integrity of the plasma membrane and thus avoiding

Apoptosis (or “programmed cell death”) is a natural,
orderly and energy-dependent process that is charac-
terized morphologically by cell shrinkage, chromatin
condensation and DNA fragmentation, while retaining
∗ Corresponding author. Tel.: +49 341 9715722;
fax: +49 341 9715709.
E-mail address: Beate.Fuchs@medizin.uni-leipzig.de (B. Fuchs).
an inflammatory response. Apoptosis is a physiologi-
cally important process during both tissue development
and regeneration (Meier et al., 2000), where it can be trig-
gered either by a decrease in survival factors (Williams
et al., 1990) or by an increase in factors which cause cell
damage (Rich et al., 2000). In either case, the apoptotic
response is triggered by an intracellular proteolytic cas-
cade (Hengartner, 2000) and accompanied by changes
in the glycerophospholipid (GPL) composition of the
outer surface of the cell membrane, loss of membrane

0009-3084/$ – see front matter © 2007 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.chemphyslip.2007.08.005
230 B. Fuchs et al. / Chemistry and Physics of Lipids 150 (2007) 229–238
asymmetry (negatively charged phosphatidylserine is
transferred from the inner to the outer leaflet), and for-
mation of lipid-enclosed vesicles.
The relevance of apoptosis to tissue homeostasis is
exemplified by the hematopoietic system, in which pro-
genitor cells are continually produced in excess, but
depend on the presence of lineage-specific growth fac-
tors for their continued survival and proliferation. In
this way, blood cell production can be trimmed by
apoptosis to match requirements (Cowling and Dexter,
1994). The multipotent progenitor Factor Dependent
Cells Paterson-Mixed potential (FDCPmix) cell lines
(Spooncer et al., 1993) reproduce this phenomenon in
vitro by demonstrating a rapid and reproducible apopto-
sis upon deprivation of interleukin 3 (IL-3) (Williams et
al., 1990).
Compared with intensively characterized cytoplas-
mic and nuclear events associated with apoptosis, the
changes in glycerophospholipid composition remain rel-
atively poorly understood, due in part to analytical
problems. The most common approach of monitor-
ing alterations in cell glycerophospholipids has usually
involved the use of radioactive labeling (Atsumi et al.,
1998). Although very sensitive, this approach is labo-
rious and time-consuming. More importantly, it would
appear that the trafficking of exogenously provided,
labeled metabolites through metabolic pools may not
necessarily reflect non-manipulated metabolic activity
(Chilton and Connell, 1988; Capriotti et al., 1988).
Additionally, Kuwae et al. (1987, 1990) employed a
stable isotope technique based on the incorporation
of 18 O from H2 18 O-containing media combined with
subsequent thin-layer chromatography (TLC) and gas
chromatography/mass spectrometry (MS) analysis. This
technique provides an indication of acyl turnover via free
fatty acids, but effectively ignores transacylations, since
18 O is not involved in this reaction.
High-resolution 31 P NMR was first applied to the
analysis of apoptosis by Tome et al. (2003), who used
a model in which a highly transformed tumor cell line
was induced to undergo apoptosis in response to glu-
cocorticoids. The advantages of the 31 P NMR approach
include the lack of any requirement for potentially mis-
leading labeled substrates, high-resolution detection of
single GPL species even in relatively complex mixtures
(since only a limited number of P-containing resonances
need be assigned) and the lack of solvent signals, which
avoids any requirement for their selective suppression.
Theoretically, 31 P NMR should permit distinction
between GPLs based not only on headgroup, but also
on linkage type (acyl-acyl, alkyl-acyl- or alkenyl-acyl)
and acyl chain composition (Pearce and Komoroski,
2000). In practice, however, although the differentia-
tion between saturated, moderately unsaturated (18:1 or
18:2) and highly unsaturated (20:4 or 22:6) acyl residues
is possible, detailed acyl residue analysis fails (Schiller
et al., in press). NMR is therefore not the method of
choice to monitor differences in the acyl chain com-
positions of GPL. However, mass spectrometry, and in
particular matrix-assisted laser desorption and ionization
time-of-flight (MALDI-TOF) MS, is emerging as a par-
ticularly powerful analytical tool in this respect (Schiller
and Arnold, 2000; Schiller et al., 2004, 2007).
We have therefore combined 31 P NMR and
MALDI-TOF MS as complementary analytical tools,
and have previously shown that the determina-
tion of the phosphatidylcholine/lysophosphatidylcholine
(PC/LPC) ratio by 31 P NMR spectroscopy and MALDI-
TOF MS provides a reliable measure of metabolic
changes in GPL composition (Fuchs et al., 2005) and in
phospholipase A2 (PLA2 ) activity (Petković et al., 2002).
Both techniques may be carried out without altering the
sample composition by adding internal standards. While
this excludes absolute quantitative analyses, calcula-
tions based on relative peak intensities yield reasonable
correlations between data obtained by 31 P NMR and
MALDI-TOF MS (Arnhold et al., 2002; Estrada and
Yappert, 2004).
We report here the combined 31 P NMR and MALDI-
TOF MS analysis of hematopoietic progenitor cells
undergoing apoptosis under the physiologically relevant
conditions of growth factor withdrawal. This combi-
nation of methods revealed a previously undescribed
decrease in diacyl-PC and -phosphatidylethanolamine
(PE) species with a concomitant increase in ether-
linked glycerophosphocholines and -ethanolamines in
the membranes of cells undergoing apoptosis. In addition
to describing relevant changes in glycerophospholipids
during apoptosis, the present study suggests that the
combination of MALDI-TOF MS techniques with 31 P
NMR may be generally applicable to high-resolution
metabolic studies.
2. Materials and methods
2.1. Chemicals
All chemicals for NMR spectroscopy (sodium
cholate, EDTA, and deuterated water with an isotopic
purity of 99.6%), buffer preparation (NaCl, TRIS) and
mass spectrometry (para-nitroaniline (PNA), CsCl) as
well as all solvents (chloroform and methanol), PLA2
from hog pancreas, trypan blue and propidium iodide
(PI) were obtained in the highest commercially available
 B. Fuchs et al. / Chemistry and Physics of Lipids 150 (2007) 229–238
purity from Fluka Feinchemikalien GmbH (Taufkirchen,
Germany). GPL used as standards were purchased as
10 mg/ml solutions in CHCl3 from Avanti Polar Lipids
(Alabaster, MA, USA). Iscove’s Modified Dulbecco’s
Medium (IMDM) was purchased from Gibco BRL®
(Invitrogen) (Carlsbad, California, USA); IL-3 was from
R&D Systems (Minneapolis, Minnesota, USA) and
horse serum from Sigma (Munich, Germany). Annexin
V (bound to the fluorophore phycoerythrin) was pur-
chased from BD (Becton Dickinson) Biosciences (USA).
2.2. Cell culture and lipid extraction
A total of 4 × 108 FDCPmix cells were grown accord-
ing to Spooncer et al. (1986) and Heyworth et al.
(1990) in IMDM containing 7% mouse IL-3-conditioned
medium (corresponding to 100 U recombinant IL-3 per
ml) and 20% horse serum. 1 × 108 cells were harvested
as a control sample (time point: 0 h). The remaining
3 × 108 cells were then washed twice with phosphate-
buffered saline to remove all traces of IL-3 containing
medium, and split between three identical cultures at
5 × 105 cells per ml in medium without IL-3. One culture
was then harvested at each time point of 8, 16 and 24 h
following IL-3 deprivation and the number of dead and
apoptotic cells determined by trypan blue staining and
annexin V binding, respectively. To evaluate the repro-
ducibility of the data, all samples were investigated in
triplicate.
Cells were harvested by centrifugation and washed
twice with 0.9% NaCl solution to remove medium con-
stituents. Previous experiments have shown that under
these conditions no loss of PL occurs if the cell mem-
branes are intact (Belgacem et al., 2007). The lipid
extraction was performed according to Folch et al.
(1957) by resuspension of the cell pellets in 0.9%
NaCl solution and the subsequent addition of chloro-
form/methanol (2:1:1, chloroform:methanol:water, final
volume ratio). Aliquots of the corresponding culture
media (supernatants) were extracted in the same man-
ner for comparison, although the majority of the
lipids present in the culture medium are expected
to stem from the supplemented horse serum, which
contains predominantly phosphatidylcholine (PC),
sphingomyelin (SM) and lyso-PC (Belgacem et al.,
2007).
After separation of the chloroform and the aqueous
phases, each was evaporated to dryness in a centrifu-
gal evaporator (Jouan, Germany). The precipitate at the
interphase between the organic and aqueous layer was
discarded and not further investigated, so that lipids
tightly bound to proteins were excluded from the study.
231
2.3. Apoptosis assay
In order to follow the progression of apoptosis,
FDCPmix cells were incubated in IL-3 deficient medium
for 0, 8, 16 and 24 h. Harvested cells were washed
twice with phosphate-buffered saline and analyzed
by phycoerythrin-labeled annexin V/propidium iodide
staining, which allows the discrimination of viable
(PI− , Annexin− ), apoptotic (PI− , Annexin+ ), and dead
(PI+ , Annexin+ ) cells (Van Engeland et al., 1998).
This assay was used according to the manufacturers
instructions. Briefly, 1 × 105 cells were stained with
5 ␮l phycoerythrin-labeled annexin V and 2 ␮g/ml PI
in 100 ␮l staining buffer (140 mM NaCl; 10 mM Hepes,
pH 7.4; 2.5 mM CaCl2 ) for 15 min at room temper-
ature. Cells were then diluted with 400 ␮l staining
buffer and kept on ice until flow cytometric analysis.
A LSR II flow cytometer (Becton Dickinson) equipped
with one blue (488 nm) and one UV (350 nm) laser
allowed the simultaneous analysis of phycoerythrin-
labeled annexin V and PI in separate fluorescence
channels.
2.4. GPL analysis by 31 P NMR spectroscopy
The dried organic and aqueous phases of the cellu-
lar extracts were solubilized according to Pearce and
Komoroski (2000) and London and Feigenson (1979)
in 50 mM TRIS (pH 7.65) containing 200 mM sodium
cholate and 5 mM EDTA. After vortexing, 31 P NMR
spectra were recorded on 0.5 ml samples in 5 mm NMR
tubes on a Bruker DRX-600 spectrometer operating
at 242.88 MHz for 31 P. All measurements were per-
formed using a direct “broadband” NMR probe at 37 ◦ C
with composite pulse decoupling (Waltz-16) to elimi-
nate 31 P–1 H coupling. Pulse intervals of the order of T1
were used to allow quantitative analysis of GPL integral
intensities (Schiller and Arnold, 2002).
Other NMR parameters were as follows. Acquisi-
tion time: 1 s, data size: 8–16 k, 60◦ pulse (7 ␮s), pulse
delay 2 s and a line-broadening (LB) of 1 Hz. Chemical
shifts were referenced to the most intense resonance of
diacyl-PC at δ = −0.6 ppm. All peak assignments were
confirmed by comparison with the shift of commercially
available GPL samples. Some assignment problems
could further be solved by the digestion of the obtained
cellular extracts with the enzyme PLA2 , which selec-
tively cleaves the acyl chain in sn-2 position (Schiller et
al., 2003). This approach was also useful in the context
of MALDI-TOF MS. No internal standard was added.
Spectra were processed using the software “1D WIN-
NMR” version 6.2® (Bruker Analytische Messtechnik
232 B. Fuchs et al. / Chemistry and Physics of Lipids 150 (2007) 229–238
Table 1
The accumulation of apoptotic and dead FDCPmix cells following IL-3 withdrawal
Time Trypan blue exclusion
point (h) (viability) (%)
0 100
8 100
16 63
24 29
Trypan blue exclusion (column 2) shows that dead cells begin to accumulate between 8 and 16 h following withdrawal. The annexin V/propidium
iodide procedure leads to the loss of most of these damaged cells (compare columns 2 and 3), but reveals a steady accumulation of apoptotic cells
in the remaining population beginning within 8 h after IL-3 withdrawal.
GmbH, Rheinstetten) including the deconvolution (II)
routine for peak area determination. Integral intensi-
ties of selected phosphorus-containing metabolites were
divided by the total areas of all detected peaks. All cal-
culations therefore involve relative rather than absolute
quantitation.
2.5. GPL analysis by MALDI-TOF MS
Positive and negative ion MALDI-TOF mass spectra
were acquired on a Bruker Daltonics Autoflex worksta-
tion (Bruker, Germany). The system utilizes a pulsed
nitrogen laser emitting at 337 nm. The extraction volt-
age was 20 kV, the “low mass gate” was turned on
at m/z = 400 and 128 single laser shots were averaged
for each mass spectrum. In order to enhance the spec-
tral resolution, spectra were recorded in the reflector
mode under “delayed extraction” conditions (Schiller
and Arnold, 2000; Schiller et al., 2004).
Lipid extracts were applied to the MALDI target
using para-nitroaniline as matrix (Estrada and Yappert,
2004). PNA was used as a 0.17 mol/l stock solution
in CHCl3 /CH3 OH (2:1, v/v) and mixed 1:1 (v/v) with
the lipid samples of interest. One important advantage
of PNA as matrix is that phosphatidylethanolamine is
detectable under these conditions as negative ion and,
therefore, not suppressed by intense positive ion PC
signals (Estrada and Yappert, 2004). CsCl was added
in selected cases to overcome the problem of overlap
between H+ and Na+ adducts and differences in the acyl
chain compositions in the positive ion spectra of the
organic extracts of FDCPmix cells (Schiller et al., 1999,
2001). Peak assignments were further confirmed by the
digestion of FDCPmix cells with pancreatic PLA2 . All
spectra were processed using the software “Flex Analy-
sis” version 2.2 (Bruker Daltonics, Germany). Positional
analysis of fatty acyl residues was further confirmed
by post source decay (PSD) experiments (Fuchs et al.,
2007).
Annexin V dead Annexin V apoptotic Annexin V
cells (%) cells (%) vital cells (%)
1.1 3.5 95.4
1.3 15.6 83.1
9.7 32.6 57.7
10.3 41.3 48.4
3. Results
Consistent with published observations (Williams et
al., 1990) the FDCPmix cells deprived of IL-3 underwent
rapid apoptosis, with 100% viability (determined by try-
pan blue exclusion) at both 0 and 8 h, 63% viability after
16 h and only 29% viability after 24 h. Complementary
data concerning apoptosis were determined by annexin
V/propidium iodide staining of the cells in the pellet sub-
sequent to centrifugation and resuspension. The trypan
blue exclusion and annexin V/propidium iodide stain-
ing results at each time point are shown in Table 1.
Trypan blue staining identifies dead cells with dam-
aged membranes without differentiating between vital
and apoptotic cells, while annexin V/propidium iodide
staining differentiates between the vital and apoptotic
cells, but involves harvesting steps which lead to the loss
of many membrane-damaged dead cells. Following IL-
3 withdrawal apoptotic cells are already detected after
8 h. In contrast, dead cells (determined by trypan blue
exclusion) are detected only after 16 h and their number
increases significantly between 16 and 24 h. Only viable
(including apoptotic but only small numbers of dead)
cells were used in this study.
Fig. 1 shows the 31 P NMR spectra of the organic
(left) and the corresponding aqueous (right) phases of
the extracts prepared from FDCPmix cells at various
times following IL-3 withdrawal. In the 31 P NMR spec-
tra of the organic phase there was an obvious decrease
in the relative amounts of the diacyl-PC (δ = −0.60
for highly unsaturated and −0.57 ppm for moderately
unsaturated sn-2 acyl residues) and PE (δ = −0.03 ppm)
moieties accompanied by an increase of the ether-
linked glycerophosphocholines (GPCether ) (δ = −0.53
highly unsaturated and −0.50 ppm for moderately
unsaturated sn-2 acyl residues) and ether-linked glyc-
erophosphoethanolamines (GPEether ) (δ = −0.01 ppm)
with increasing time following IL-3 withdrawal. Inter-
estingly, the total diacyl-PE/diacyl-PC ratio increased
 B. Fuchs et al. / Chemistry and Physics of Lipids 150 (2007) 229–238 233

Fig. 1. 242.88 MHz 31 P NMR spectra of the organic (left) and aqueous phase (right) of FDCPmix stem cell extracts of 1 × 108 cells after proliferation
in medium containing IL-3 (a) and after 8 (b), 16 (c), and 24 h (d) subsequent to the withdrawal of IL-3. Spectra were scaled according to the intensity of
the most intense resonance and the inorganic phosphate resonance (right) was truncated for means of clarity. Note that the same number of transients
was accumulated in all experiments. The decreasing signal-to-noise ratio from (a) to (d) stems from the increased number of dead cells in the
culture which are in the main part not included in the harvested cell pellets (cf. Table 1). Abbreviations: DHSM, dihydrosphingomyelin; diacyl-PC,
diacyl-phosphatidylcholine; diacyl-PE, diacyl-phosphatidylethanolamine; GPCether , alkyl- or alkenyl-acyl-glycerophosphocholine; GPEether , alkyl-
or alkenyl-acyl-glycerophosphoethanolamine; GPC, glycerophosphocholine; GPE, glycerophosphoethanolamine; LPA, lysophosphatidic acid; LPC,
lysophosphatidylcholine; LPE, lysophosphatidylethanolamine; PG, phosphatidylglycerol; PI, phosphatidylinositol; PS, phosphatidylserine; SM,
sphingomyelin.

progressively over this period from 31% (0 h) to 37%
(8 h) and 44% (16, 24 h). Despite the obvious degra-
dation of diacyl-PC and PE, only minor amounts of
LPC (δ = −0.14 ppm) and LPE (δ = 0.47 ppm) reso-
nances were detectable in cell extracts even after 24 h.
Analysis of the aqueous phase (Fig. 1, right) of the
FDCPmix cell extracts revealed that the glycerophos-
phocholine (GPC) peak reached a maximum at 8 h,
while phosphocholine decreased continuously with the
progression of apoptosis and was barely detectable
after 24 h. In contrast, the lysophosphatidic acid (LPA)
content remained constant. A weak resonance of glyc-
erophosphoethanolamine (GPE), which represents a
breakdown product of the PE metabolism, was detectable
by 8 h but remained at a very low level for the remaining
time points. The decrease in the signal-to-noise ratio of
the 31 P NMR spectra throughout the course of the exper-
iment is a consequence of reduced cell yield from the
later time points (dead cells with permeable membranes
being removed by the washing steps).
Fig. 2 shows positive (left) and negative ion (right)
MALDI-TOF mass spectra of the organic phases of the
234 B. Fuchs et al. / Chemistry and Physics of Lipids 150 (2007) 229–238

Fig. 2. Positive (left) and negative (right) ion MALDI-TOF mass spectra of the organic phase of FDCPmix stem cells after proliferation in medium
containing IL-3 (a) and subsequent to the withdrawal of IL-3 after 24 h (b). Dotted lines mark the selected peaks used for quantitative analysis
as shown in (c). The ratios prior to the IL-3 withdrawal (0 h) are indicated by white bars; those at 8 h following withdrawal are marked by light
gray, after 16 h by dark gray, and after 24 h by black bars. Relative intensity ratios of GPCether 16:0, 18:1 (m/z = 878.5) and either PC 16:0, 18:2
(m/z = 890.5) or PC 16:0, 18:1 (m/z = 892.5) or PC 16:0, 20:4 (m/z = 914.5) or PC 18:0, 20:4 (m/z = 942.5) as well as relative intensity ratios of
GPEether 18:0, 20:5 and PE 18:0, 20:4 (m/z = 750.5/766.5) are given. Please note that the Cs+ adducts were used exclusively for the analysis of the
positive ion spectra. Error bars represent standard deviations of three independent measurements from three different cell cultures. Note that the
acyl chain positions of the selected GPLs were determined by additional post source decay (PSD) MS experiments. For further details see Schiller
et al. (2007) and Fuchs et al. (2007).
 B. Fuchs et al. / Chemistry and Physics of Lipids 150 (2007) 229–238 235

Table 2
Assignments of the positive and negative ion signals obtained from the MALDI-TOF mass spectra of the organic extracts of FDCPmix cells
Mass m/z Assignment Mass m/z Assignment
positive mode positive mode

850.5 [Alkyl-GPC 32:1 or alkenyl-GPC 32:0 + Cs]+ 918.5 [Diacyl-PC 36:2 + Cs]+
852.5 [Alkyl-GPC 32:0 + Cs]+ 920.5 [Diacyl-PC 36:1 + Cs]+
862.5 [Diacyl-PC 32:2 + Cs]+ 924.5 [Alkyl-GPC 38:6 or alkenyl-GPC 38:5 + Cs]+
864.5 [Diacyl-PC 32:1 + Cs]+ 926.5 [Alkyl-GPC 38:5 or alkenyl-GPC 38:4 + Cs]+
866.5 [Diacyl-PC 32:0 + Cs]+ 928.5 [Alkyl-GPC 38:4 or alkenyl-GPC 38:3 + Cs]+
876.5 [Alkyl-GPC 34:2 or alkenyl-GPC 34:1 + Cs]+ 932.5 [Alkyl-GPC 38:2 or alkenyl-GPC 38:1 + Cs]+
878.5 [Alkyl-GPC 34:1 or alkenyl-GPC 34:0 + Cs]+ 938.5 [Diacyl-PC 38:6 + Cs]+
880.5 [Alkyl-GPC 34:0 + Cs]+ 940.5 [Diacyl-PC 38:5 + Cs]+
888.5 [Diacyl-PC 34:3 + Cs]+ 942.5 [Diacyl-PC 36:4 + Cs]+
890.5 [Diacyl-PC 34:2 + Cs]+ Negative mode
892.5 [Diacyl-PC 34:1 + Cs]+ 722.5 [Alkyl-GPE 36:5 or alkenyl-GPE 36:4 − H]−
898.5 [Alkyl-GPC 36:5 or alkenyl-GPC 36:4 + Cs]+ 726.5 [Alkyl-GPE 36:3 or alkenyl-GPE 36:2 − H]−
900.5 [Alkyl-GPC 36:4 or alkenyl-GPC 36:3 + Cs]+ 728.5 [Alkyl-GPE 36:2 or alkenyl-GPE 36:1 − H]−
902.5 [Alkyl-GPC 36:3 or alkenyl-GPC 36:2 + Cs]+ 742.5 [Diacyl-PE 36:2 − H]−
904.5 [Alkyl-GPC 36:2 or alkenyl-GPC 36:1 + Cs]+ 744.5 [Diacyl-PE 36:1 − H]−
906.5 [Alkyl-GPC 36:1 or alkenyl-GPC 36:0 + Cs]+ 748.5 [Alkyl-GPE 38:6 or alkenyl-GPE 38:5 − H]−
912.5 [Diacyl-PC 36:5 + Cs]+ 750.5 [Alkyl-GPE 38:5 or alkenyl-GPE 38:4 − H]−
914.5 [Diacyl-PC 36:4 + Cs]+ 764.5 [Diacyl-PE 38:5 − H]−
916.5 [Diacyl-PC 36:3 + Cs]+ 766.5 [Diacyl-PE 38:4 − H]−

Spectra were recorded in the presence of an excess of CsCl. The number of carbon atoms and double bonds in both individual acyl chains were
combined. It should be noted that even if PSD spectra were recorded in all cases, assignment was sometimes equivocal. This was particularly true
for the alkyl-acyl and alkenyl-acyl differentiation.

same FDCPmix cell extracts in the presence of IL-3 (a)
and after 24 h of IL-3 withdrawal (b). Positive ion spec-
tra were recorded in the presence of CsCl in order to
avoid overlap between the different adducts and differ-
ences in acyl chain compositions (Schiller et al., 2001).
Accordingly, all masses are shifted by 132.9 Da to higher
masses. The signal-to-noise ratio is slightly lower in
the negative ion mass spectra because of its lower ion-
ization efficiency (Schiller et al., 2004). Although the
entire spectrum was analyzed, Fig. 2 focuses on those
mass regions exhibiting the most pronounced changes
in GPL composition, i.e. the ether-linked glycerophos-
phocholines and diacyl-PCs in the positive ion mode
and the ether-linked glycerophosphoethanolamines and
diacyl-PEs in the negative ion mode. All m/z values
were assigned according to Table 2. The MALDI-TOF
mass spectra clearly confirm the results obtained by 31 P
NMR: the ratio between the ether-linked glycerophos-
phocholines and the diacyl-PC as well as that between
the ether-linked glycerophosphoethanolamines and the
diacyl-PE increases with time, i.e. with the proportion
of apoptotic cells.
In addition to the changes in the relative amounts of
head groups and linkage types, the MALDI-TOF mass
spectrometric analysis highlights changes of the acyl
chain compositions of the individual glycerophospho-

Fig. 3. Relative contribution of phosphorus-containing metabolites (as percentage of total phosphorus) determined by 31 P NMR from the organic
extracts (a) and the corresponding organic extracts of the cell culture medium (b) of FDCPmix cells. The values prior to the IL-3 withdrawal (0 h)
are indicated by white bars; those at 8 h following withdrawal are indicated by light gray, after 16 h by dark gray, and after 24 h by black bars. Error
bars represent standard deviations of three independent measurements from three different cell cultures.
236 B. Fuchs et al. / Chemistry and Physics of Lipids 150 (2007) 229–238
lipids. Specifically, the comparison of the intensities of
diacyl-PC 16:0/18:2 (m/z = 890.5), diacyl-PC 16:0/20:4
(m/z = 914.5) and diacyl-PC 18:0/20:4 (m/z = 942.5)
with the intensity of diacyl-PC 16:0/18:1 (m/z = 892.5),
revealed that the first three compounds are metabolized
to a higher extent in apoptotic cells. A similar bias in
degradation pattern is seen in the corresponding diacyl-
PE derivatives (summarized in Fig. 2c). This suggests
that more highly unsaturated glycerophospholipids are
preferentially metabolized during apoptosis.
More quantitative comparisons of the changes in
glycerophospholipid composition during apoptosis were
obtained by integration (Fig. 3) of the individual 31 P
NMR resonances shown in Fig. 1. From trace (3a) it is
evident that the moiety of diacyl-PC and PE decreased,
whereas the moiety of ether-linked glycerophospho-
cholines and ethanolamines increased during apoptosis.
The most significant change in the bulk phospholipids
(e.g. the diacyl-PC content) occurred over the first 16 h
and was only slightly changed at later time points.
This correlates closely with the accumulation of apop-
totic cells (cf. Table 1). The progressive degradation of
diacyl-PC during apoptosis would be expected to result
in LPC generation which must either accumulate in
the apoptotic cells themselves or could be released to
the supernatant. Although a very small LPC resonance
appeared in the cell extracts by 24 h of IL-3 withdrawal
(Fig. 1) its absolute level was very low. In contrast, we
detected a clear accumulation of LPC in the supernatant
(trace 3b). The potential contribution of released PLA2
(which could theoretically generate LPC from serum
constituents present in the growth medium) could be
ruled out by the lack of detectable degradation of PC
22:1/22:1 added to the medium as an artificial lipid that
does not occur in living organisms (data not shown).
We therefore conclude that the degradation of diacyl-
PC in this cellular system leads to the early release of
most of the LPC to the supernatant rather than reten-
tion in the apoptotic cells. As we have not investigated
the precipitated protein layer at the interphase between
aqueous and organic layer, a partial loss of LPC due
to binding to the precipitated protein cannot be ruled
out.
4. Discussion
We report here the combined use of high-resolution
31 P NMR spectroscopy and MALDI-TOF MS to moni-
tor changes in the principal glycerophospholipid classes
and their metabolites in hematopoietic progenitor cells
undergoing the physiologically relevant process of apop-
tosis in response to growth factor deprivation (Williams
et al., 1990), with the focus on ether-linked and diacyl-
glycerophospholipids. The number of dead cells in the
cell culture was determined by trypan blue exclusion
and the number of dead and apoptotic cells subsequent
to washing of the cell pellet by the annexin V/propidium
iodide assay (Van Engeland et al., 1998). While 31 P
NMR provides a direct quantitative measurement of
species separable by chemical shift differences mostly
within the head groups, MALDI-TOF MS is inherently
less quantitative (Schiller et al., 2004), but has high
resolving power for PL species differing in acyl chain
composition. The data provided in this study are rel-
ative rather than absolute firstly because the amount
of extractable lipids depends significantly on the sol-
vent mixture used and secondly because we cannot
rule out the loss of some lipids in the precipitated
protein layer between the organic and the aqueous
phase. It has been shown by others that these prob-
lems can be overcome by “dissolving” the cells directly
in the detergent used for the preparation of the NMR
samples (Nouri-Sorkhabi et al., 1996). However, using
these conditions we observed severe line-broadening
of 31 P resonances (data not shown), making assign-
ments ambiguous and therefore chose not to use this
approach.
Both methods (31 P NMR and MALDI-TOF MS)
revealed an apoptosis-associated increase in the rel-
ative content of ether-linked glycerophospholipids
and a decrease in the relative content of diacyl-
glycerophospholipids during apoptosis. The extent and
time-dependence of the observed bulk phospholipids
correlates approximately with the relative content of
apoptotic cells but not of dead cells. Additionally, the
MALDI-TOF MS data indicate that the metabolic fate
of glycerophospholipids depends on their acyl chain
composition. The increase in ether-linked glycerophos-
pholipids suggests that they may serve as a reservoir
for arachidonoyl residues released by the breakdown
of diacyl-glycerophospholipids, thereby helping to
prevent further metabolism of arachidonic acid to pro-
inflammatory prostaglandins. At the same time, the early
release of the remaining LPC moiety to the medium may
also be necessary to preserve the membrane integrity
characteristic of the apoptosis. Although LPC may be
further metabolized to GPC and free fatty acids, only
GPC was monitored in this study because free fatty acids
can be detected unambiguously neither by 31 P NMR nor
by MALDI-TOF MS (Schiller et al., 2004). For these
reasons, we did not try to clarify the metabolic fate of
the LPC in more detail.
Diacyl-PC is generated in healthy cells by de novo
biosynthesis through a cascade of three enzymatic
 B. Fuchs et al. / Chemistry and Physics of Lipids 150 (2007) 229–238
steps from choline to phosphocholine conversion and
finally to PC condensation (Kennedy pathway) or
by a remodeling process mediated by at least two
mechanisms: a deacylation–reacylation process or a
deacylation–transacylation reaction (Podo, 1999). Ether-
linked glycerophosphocholines follow a distinct de novo
biosynthesis pathway (Nagan and Zoeller, 2001). Our
results are consistent with those of a previous study,
which concluded that ether-linked glycerophospholipids
have lower turnover rates than the corresponding di-
acyl species (Kuwae et al., 1990). Another possible
mechanism involves a transacylation process of the
sn-2 acyl group from diacyl- to ether-linked glyc-
erophosphocholines (Kuwae et al., 1990). Regardless
of the synthesis pathway of ether-linked glycerophos-
pholipids recent studies on structure, dynamics, and
conformation have demonstrated significant differences
between ether-linked and diacyl-glycerophospholipids
associated with the sn-1 ether linkage (Ford and Gross,
1994): Ether-linked glycerophosphocholine membrane
bilayers are far more densely packed than diacyl-
glycerophospholipids (Ford and Gross, 1994). Further-
more, the substantially different molecular geometry
compared to diacyl-glycerophospholipids influences
their ability to modulate the kinetics of ion transport
(Chen and Gross, 1994). In this respect, it is important
to note that ionic homeostasis is essential for the normal
physiology of all cell types, and that the deregulation of
ionic homeostasis is a common hallmark of apoptosis,
with the depolarization of plasma membranes occurring
as an early feature of apoptosis (Franco et al., 2006). Our
data are therefore closely consistent both with previous
findings and with the known physiological characteris-
tics of apoptosis.
In conclusion, the combined application of 31 P NMR
and MALDI-TOF MS to FDCPmix cell extracts has
revealed evidence for significant changes in membrane
glycerophospholipid composition during the apoptosis
of hematopoietic progenitor cells. The combination of
these techniques clearly reveals relevant changes with-
out the requirement for isotope or other labeling that
might influence the metabolic processes under study.
In addition to describing relevant changes in glyc-
erophospholipids during apoptosis, the present study
therefore suggests that the combination of MALDI-
TOF MS techniques (developed primarily for proteomics
studies) with 31 P NMR may be generally advanta-
geous in high-resolution metabolic studies. In pursuit
of this aim, we are currently focusing on approaches
to making MALDI-TOF MS capable of providing
absolute rather than relative data on GPL composi-
tion.
237
Acknowledgements
This work was supported by the Deutsche
Forschungsgemeinschaft (DFG CR132/3 and Schi
476/5-1) and the German Ministry of Education and
Research (BMBF Grants 0313836 and 0313833A).
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