A simple method to generate oxidized phosphatidylcholines in amounts close to one milligram

Article abstract of DOI:10.1016/j.chemphyslip.2014.09.003

Oxidized (phospho)lipids are of paramount interest from different reasons: beside their significant in vivo relevance, these products are often needed in the laboratory to study the response of selected cells to oxidized lipids. Unfortunately, the commercial availability of oxidized lipids is limited and scientists interested in studying the physiological impact of oxidized lipids are normally forced to prepare the required compounds by themselves. We will show here that chain-shortened products of oxidized phosphatidylcholines (PCs) such as aldehydes and carboxylic acids can be easily (and in nearly quantitative yields) generated by the Fenton reaction (H₂O₂ + Fe²⁺) or the KMnO₄-induced oxidation of the PC. Using the Fenton reaction and physiological saline, chlorinated oxidation products such as chlorohydrins are also readily available. Additionally, it will be shown that preparative thin-layer chromatography (TLC) is a convenient but simple method to isolate the individual oxidation products in reasonable yields and high purities: all relevant products could be successfully identified by matrix-assisted laser desorption and ionization (MALDI) mass spectrometry and the amounts of the oxidized products determined by a simple colorimetric assay.

Full text of DOI:10.1016/j.chemphyslip.2014.09.003

Chemistry and Physics of Lipids 184 (2014) 30–37
Contents lists available at ScienceDirect
Chemistry and Physics of Lipids
journal homepage: www.elsevier.com/locate/chemphyslip
Short communication
A simple method to generate oxidized phosphatidylcholines in
amounts close to one milligram
*
Kristin Zschörnig, Jürgen Schiller
University of Leipzig, Faculty of Medicine, Institute of Medical Physics and Biophysics, Härtelstr. 16/18, D-04107 Leipzig, Germany
A R T I C L E I N F O
A B S T R A C T
Article history:
Received 5 May 2014
Received in revised form 27 August 2014
Accepted 15 September 2014
Available online 18 September 2014
Oxidized (phospho)lipids are of paramount interest from different reasons: beside their significant in vivo
relevance, these products are often needed in the laboratory to study the response of selected cells to
oxidized lipids. Unfortunately, the commercial availability of oxidized lipids is limited and scientists
interested in studying the physiological impact of oxidized lipids are normally forced to prepare the
required compounds by themselves.
We will show here that chain-shortened products of oxidized phosphatidylcholines (PCs) such as
aldehydes and carboxylic acids can be easily (and in nearly quantitative yields) generated by the Fenton
reaction (H2O2 + Fe2+) or the KMnO4-induced oxidation of the PC. Using the Fenton reaction and
physiological saline, chlorinated oxidation products such as chlorohydrins are also readily available.
Additionally, it will be shown that preparative thin-layer chromatography (TLC) is a convenient but
simple method to isolate the individual oxidation products in reasonable yields and high purities: all
relevant products could be successfully identified by matrix-assisted laser desorption and ionization
(MALDI) mass spectrometry and the amounts of the oxidized products determined by a simple
colorimetric assay.
Keywords:
Phosphatidylcholine
Lipid (per)oxidation
Thin-layer chromatography
MALDI MS
ã 2014 Elsevier Ireland Ltd. All rights reserved.
1. Introduction
and Parkinson’s disease (Mattson, 2009). Finally, selected oxidized
PLs (for instance, oxidatively-modified phosphatidylcholines and
Phospholipids (PLs) are (beside cholesterol and selected
membrane proteins) important constituents of all cellular
membranes. Because of their amphiphilic properties PLs enable
the formation of stable bilayers that protect the interior of the cells
but enable also selective transport processes. Additionally, selected
PLs such as phosphatidic acids or poly-phosphoinositides are
molecules with important regulatory functions serving as lipid
second messengers (Martelli et al., 2004).
The oxidation of PLs by reactive oxygen species (ROS) is a very
important reaction and many oxidized PLs are of considerable
biological significance (Fuchs et al., 2011). This particularly applies
at inflammatory conditions that are accompanied by increased ROS
generation by immune cells such as neutrophilic granulocytes.
Lipid oxidation is involved in the pathogenesis of many diseases
such as asthma (Olopade et al.,1997), rheumatoid arthritis (Schiller
et al., 2003; Fuchs et al., 2005), atherosclerosis (Victor et al., 2009)
lyso-phosphatidylcholines) are known to possess biological
activities similar to platelet-activating factors (PAF) (Marathe
et al., 2000) and are likely to destabilize the structures of biological
membranes.
Phosphatidylcholines (PCs) are the most abundant PLs in the
human body as well as in virtually all mammalian cells (Henry
et al., 1998). This – in combination with their commercial
availability – renders PCs interesting compounds to study their
oxidation products. The related oxidation reactions are not only of
interest regarding mechanistic considerations (e.g., to investigate
the stabilities of the oxidation products) but as well to study the
response of selected cells towards selected oxidation products.
Unfortunately, oxidized (phospho)lipids are scarcely commercially
available and, thus, scientists are often forced to synthesize the
desired oxidation products by themselves.
The oxidation of unsaturated PCs such as PLPC (1-palmitoyl-2-
linoleoyl-sn-phosphatidylcholine) leads to per-oxidized com-
pounds (endo- and hydroperoxides) as primary products which
decay subsequently under generation of aldehydes as well as
carboxylic acids as secondary products, i.e., a scission of the double
bonds occurs (Niki, 2009; Fuchs et al., 2011). Although oxidation
reactions are commonly used to determine the positions of double
* Corresponding author at: Universität Leipzig, Medizinische Fakultät Institut für
Medizinische Physik und Biophysik, Härtelstr. 16/18, D-04107 Leipzig, Germany.
Tel.: +49 341 9715733; fax: +49 341 9715709.
E-mail address: juergen.schiller@medizin.uni-leipzig.de (J. Schiller).
http://dx.doi.org/10.1016/j.chemphyslip.2014.09.003
0009-3084/ã 2014 Elsevier Ireland Ltd. All rights reserved.

K. Zschörnig, J. Schiller / Chemistry and Physics of Lipids 184 (2014) 30–37
31
bonds within an unsaturated fatty acyl chain, many details of lipid
oxidation processes remain to be elucidated (Fuchs et al., 2011).
The present work has two different aims: on the one hand,
different oxidizing agents (atmospheric oxygen, KMnO4 and the
Fenton reagent) are compared (a) regarding their ability to induce
lipid oxidation and (b) the generated product patterns. On the other
hand, preparative thin-layer chromatography (TLC) will be used to
isolate the most abundant lipid oxidation products: although the
separation quality achievable by TLC is rather poor in comparison to
HPLC (high performance liquid chromatography), the required TLC
equipment is inexpensive and simple and the method can be, thus,
easilyestablishedinvirtuallyalllaboratories.PLPCwillbeexclusively
usedherebecauseitrepresentsintheseauthor'sopinionanexcellent
compromise: due to its two double bonds (within the linoleoyl
residue) it is more readily oxidized in comparison to POPC
(1-palmitoyl-2-oleoyl-sn-phosphatidylcholine) that contains only
a single double bond. On the other hand, the variety of products is
much smaller in comparison to higher unsaturated PLs (Reis et al.,
2004).
aqueous phase (the organic phase was nearly colorless, while the
aqueous phase was violet).
2.4. Oxidation of phospholipids by the Fenton reagent (H2O2 and FeCl2)
An aliquot of 33 nmol PLPC (25
evaporated to dryness. The resulting lipid film was incubated with
a fixed amount of 50 l of a 500 mM aqueous solution of H2O2
(spectrophotometrically determined (
230 = 74 Mꢁ1 cmꢁ1)) (Beers
and Sizer, 1952) and 50 l of a 50 mM aqueous FeCl2 solution.
mg dissolved in 10 ml CHCl3) was
m
e
m
These conditions resulted in our hands in a maximum yield of
oxidation products. Incubations were performed for 1 h, 2 h or over
night (about 16 h). The incubation was stopped by adding
chloroform/methanol (1:1 (v/v)) to extract the lipids (Bligh and
Dyer, 1959) and to reduce the amounts of inorganic salts.
2.5. Large scale oxidation of PLPC with H2O2 and FeCl2
An aliquot of 1.32
was evaporated to dryness. The resulting lipid film was incubated
with a fixed amount of 50 l H2O2 (500 mM dissolved in H2O) and
50 l FeCl2 (50 mM dissolved in H2O). Incubations were performed
mmol PLPC (1 mg dissolved in 100 ml CHCl3)
The characterization of the generated oxidation products will
be performed by matrix-assisted laser desorption and ionization
(MALDI) time-of-flight (TOF) mass spectrometry (MS) (Fuchs et al.,
2010), which is a simple, but powerful method to characterize
lipids and their oxidation products. Finally, the yields of the
different oxidation products will be determined by using a simple
colorimetric assay.
m
m
over night (about 12 h). The incubation was stopped by adding
chloroform/methanol (1:1 (v/v)) to extract the lipids (Bligh and
Dyer,1959). Six different reaction batches were combined and used
for preparative TLC. The total amount of lipid was, thus, 6 mg
corresponding to about 7.9 mmol.
2. Materials and methods
2.6. Lipid extraction
2.1. Chemicals
Lipid extraction was performed in all cases according to Bligh
and Dyer (1959). After addition of the organic solvent mixtures
(aqueous phase/chloroform/methanol = 1:1:1 (v/v/v)) the sample
was vigorously vortexed and the mixture centrifuged at 1000 ꢂ g
for 5 min (296 K) to expedite the separation of the organic and the
aqueous phase. The lower (chloroform) phase was carefully
isolated by using a Hamilton syringe whereas the upper phase
(aqueous methanol) was discarded. Samples were directly used for
subsequent MALDI–TOF MS characterization or TLC separation.
All chemicals and all solvents (methanol, ethanol, chloroform,
isopropanol, acetonitrile, water, triethylamine and glacial acetic
acid) were obtained in the highest commercially available purity
from Sigma–Aldrich (Taufkirchen, Germany) and used as supplied.
PLPC was purchased from AVANTI Polar Lipids (Alabaster, AL,
USA) as 10 mg/ml chloroform solution and used as supplied.
Chemicals used for lipid oxidation were purchased either from
Fluka (KMnO4 and H2O2) or from Sigma–Aldrich (FeCl2).
The dye primuline (for monitoring the presence of lipids on the
TLC plate) was obtained from Sigma–Aldrich (Taufkirchen,
Germany) and used as previously described (Fuchs et al., 2008;
White et al.,1998). Phospholipase A2 (PLA2) from hog pancreas was
also obtained from Sigma–Aldrich.
2.7. Thin-layer chromatography (TLC)
OxidizedlipidextractswereappliedontoHPTLCsilicagel60plates
(10 ꢂ10 cm in size with aluminum backs (Merck, Darmstadt,
Germany)), using a Linomat 5 device (CAMAG; Berlin, Germany),
anddevelopedinaverticalTLCchamberwithCHCl3,methanol,water
(60:30:5 (v/v/v)) as the mobile phase (Kupke and Zeugner, 1978).
Lipids were visualized by spraying the plate with primuline (Direct
Yellow59),whichisknownto bindnon-covalentlyto theapolarfatty
acyl residues of PLs without affecting the molecular weights (White
et al., 1998). Upon illumination with UV light (366 nm), individual
lipid classes are detectable as colored spots. These spots were
assessed using a digital image system in combination with the
program Argus X1 (BioStep, Jahnsdorf, Germany).
In one selected case, two-dimensional TLC (using commercially
available diol-modified (Merck, Darmstadt, Germany) HPTLC silica
plates) was also applied in an attempt to improve the separation
quality. Chloroform, methanol, water, ethanol, triethylamine (13/4/
2/7/7, v/v/v/v/v) was used in the first dimension and (after careful
drying of the TLC plate) chloroform, methanol, acetic acid, water
(45/20/6/1, v/v/v/v) in the second dimension.
2.2. Oxidation of PLPC at room temperature (296 K)
An aliquot of about 66 nmol PLPC (50 mg, dissolved in 10 ml
CHCl3) was evaporated to dryness and the resulting lipid film
subsequently exposed to atmospheric oxygen in a small glass
vessel (the area of the lipid film exposed to oxygen was estimated
to be about 19.6 mm2). The oxidation was performed for “0”
(control), 2 and 7 days at 296 K. The incubation was stopped by the
addition of the MALDI matrix and the resulting solution
immediately analyzed by MALDI–TOF MS (vide infra).
2.3. Oxidation of PLPC by KMnO4
An aliquot of 33 nmol PLPC (25
evaporated to dryness. Oxidation was induced by the addition of
mg, dissolved in 10 ml CHCl3) was
100 ml of a 1.58 mol/l KMnO4 (250 mg/ml dissolved in H2O)
solution and incubated for 10 min at 37 ꢀC. The oxidation was
stopped by the addition of organic solvents (chloroform/methanol
2.8. Phospholipase A2 digestion
(1:1 (v/v); 200
ml)) to extract the apolar lipids from the aqueous
Selected PL oxidation mixtures were digested by the enzyme
phase, whereby the KMnO4 remains nearly quantitatively in the
PLA2 to obtain the corresponding lysolipids and to confirm peak

32
K. Zschörnig, J. Schiller / Chemistry and Physics of Lipids 184 (2014) 30–37
assignments (Fuchs et al., 2007a). Briefly, organic aliquots were
evaporated to dryness. Lipid vesicles were prepared by suspending
the resulting PL film in water and vortexing vigorously for 30 s.
Vesicles were treated with 0.5 mg/ml PLA2 (about 100 U/ml) for 2 h
at 37 ꢀC. Afterwards, lipids were extracted as described (vide supra)
and the organic solution directly used for MS characterization.
Niederau, Germany) as 10 mg/ml solution in 60/40 (v/v) iso-
propanol/acetonitrile (Sun et al., 2008). These both matrices were
used due to their different pK values (Fuchs et al., 2009). All organic
extracts were pre-mixed (1:1 (v/v)) with either 9-AA or DHB and
1 ml of this mixture was transferred onto the MALDI target (gold-
coated aluminum). All mass spectra were acquired on a Bruker
Autoflex mass spectrometer (Bruker Daltonics, Bremen, Germany).
This system utilizes a pulsed nitrogen laser emitting at 337 nm. The
extraction voltage was 20 kV and gated matrix suppression was
applied to prevent the saturation of the detector by matrix ions.
One hundred single lasers shots were averaged for each mass
spectrum. In order to enhance the resolution, all spectra were
acquired in the reflector mode using “delayed extraction”
2.9. Lipid re-extraction from silica gel
The marked lipid spots (subsequent to primuline staining) were
scratched off from the TLC plate with a steel spatula. Subsequently,
the silica gel was eluted with a 20-fold excess of 0.9% aqueous
NaCl/methanol/chloroform (1:1:1 (v/v/v)). The amount of the used
solvent is related to the dry weight of the silica gel. The silica gel
was washed three times with the indicated excess of the solvent
system to minimize lipid losses (Teuber et al., 2010). The obtained
extracts were combined and dried under reduced pressure.
According to our experience there is no major difference and
the use of an inert gas is, thus, not an absolute must.
conditions.
A more detailed methodological description of
MALDI–TOF MS with the focus on lipid analysis is available in a
recent review by Fuchs et al. (2010) as well as Bresler et al. (2011).
Raw data were processed with the software “Flex Analysis” version
2.2 (Bruker Daltonics).
3. Results and discussion
2.10. Determination of PL amounts (Stewart-assay)
The first aim of this study was to compare the effects of different
oxidizing agents on PLPC as one selected PL. The obtained
oxidation products were identified by MALDI–TOF MS which is
a simple and convenient method to characterize lipids and the
related oxidation products (Fuchs et al., 2010).
This assay developed by Stewart (1980) is a fast and simple
colorimetric method to determine absolute amounts of PLs. It is
based on the complex formation between PLs and an inorganic,
colored iron salt (ammonium ferrothiocyanate) which gets soluble
in organic solvents after binding to the PL.
A solution of ferrothiocyanate was prepared by dissolving
27.03 g FeCl3 ꢂ 6H2O and 30.4 g NH4SCN in one liter of water. The
lipid sample of interest (dissolved in 2 ml CHCl3) was added to 2 ml
ferrothiocyanate solution. After careful mixing, the organic phase
was isolated by centrifugation. The optical density of the
Fig. 1 compares the positive ion MALDI–TOF mass spectra of
PLPC subjected to different oxidizing conditions. Trace (1A) reflects
pure PLPC as the control. PLPC results in two peaks at m/
z = 758.6 and 780.6 corresponding to the H+ and the Na+ adduct
(Schiller et al., 1999). If the oxidation is performed by exposing the
PLPC to atmospheric oxygen at RT (296 K) for 1 day (trace (1B)),
there is no more original PLPC detectable. However, there are
several signals that can be easily assigned to characteristic
oxidation products: the peak at m/z = 496.3 corresponds to
lysophosphatidylcholine (LPC) 16:0 (H+ adduct) that is generated
as a minor product upon PLPC oxidation by the complete cleavage
of the unsaturated (or oxidatively modified) fatty acyl residue.
Although it is commonly accepted that LPC is primarily generated
under the influence of the enzyme phospholipase A2, oxidation
processes are also accompanied by LPC generation: Arnhold et al.
(2002) were able to show that the introduction of electronegative
chloroform phase was determined at
l= 488 nm in a quartz
cuvette on a Hitachi U-2000 photometer. Pure CHCl3 served as
blank control.
2.11. MALDI–TOF mass spectrometry
Two different matrices were applied: 2,5-dihydroxybenzoic
acid (DHB) (from Fluka) was used as 0.5 M solution in methanol
(Schiller et al., 1999) and 9-aminoacridine (9-AA) hemihydrate
(from Acros Organics, distributed by Fisher Scientific GmbH,
758.6
780.6
(A)
666.4
*
720.6
650.4
496.3
688.4
666.4
(B)
758.6
758.6
650.4
790.6
*
722.6
*
579.4
496.3
822.6
636.4
650.4
(C)
496.3
500
666.4
788.6
806.6
(D)
550
600
650
700
750
800
850
m/z [Th]
Fig. 1. Selected positive ion MALDI–TOF mass spectra of oxidized PLPC. All spectra were recorded with 9-AA as matrix and the most prominent peaks are marked by the
corresponding m/z values. (A) represents the spectrum of native PLPC (control) while the PLPC was oxidized by exposition to atmospheric oxygen (B), KMnO4 (C) and FeCl2 and
H2O2 (Fenton conditions) (D). So far unidentified peaks are marked by asterisks. Additional experimental details are available in Section 2.

K. Zschörnig, J. Schiller / Chemistry and Physics of Lipids 184 (2014) 30–37
33
Table 1
with shortened fatty acyl residues) when the oxidation is induced
by KMnO4 (trace (1C)) or the Fenton reagent (trace (1D)).
Particularly the KMnO4-induced oxidation leads to the addition
of one (m/z = 790.6) or two (m/z = 822.6) O2 molecules. Addition of
H2O2 (m/z = 792.6) to the PLPC molecule may also occur.
Unfortunately, some so far unknown products (m/z = 579.4,
720.6 and 722.6; marked by asterisks in Fig. 1) are also generated
under these experimental conditions. A more detailed assignment
of all observed peaks is available in Table 1.
In a nutshell, all three oxidation methods lead to similar
product patterns although the yields of the individual compounds
are obviously different. It is surprising that the oxidation by the
Fenton reagent resulted in the smallest yield of reaction products
because the generated hydroxyl radicals (HOꢃ) would be expected
to react in a diffusion-controlled manner. Therefore, our next step
was to evaluate the time-dependence of PLPC oxidation under
Fenton conditions: the ratios between PLPC, H2O2 and FeCl2 were
kept constant in all cases and the only variable was the incubation
time. The corresponding positive ion MALDI mass spectra are
shown in Fig. 2.
It is evident that the incubation time has a considerable impact
on the product pattern: while no oxidation products are detectable
in the spectrum of the pure PLPC (2A), peaks of LPC 16:0, the
aldehyde, the carboxylic acid and an oxidized product with
m/z = 788.6 (Davis et al., 2008; cf. Table 1) are clearly detectable
after incubation for 1 h with the Fenton reagent (2B) although the
peaks of the educt (PLPC) are still dominating. Using a prolonged
incubation time of 2 h (2C) the yield of the carboxylic acid
(m/z = 666.4 and 688.4) is significantly increased while that of the
PLPC as the educt is reduced. In addition, the peroxidized product
at m/z = 790.6 and the bis-chlorohydrin at m/z = 862.6 are detect-
able, whereas m/z = 826.6 corresponds to the loss of HCl from
m/z = 862.6. The generation of the chlorohydrins under conditions
of the Fenton reaction may be surprising but this fact was recently
established by Wu et al. (2011): these authors suggested that
chloride reacts with HOꢃ in a multiple step reaction to give HOCl
which readily adds to the unsaturated fatty acyl residues of PLs.
After 16 h (2D), the peak of the PLPC is not detectable anymore
and the carboxylic acid (m/z = 666.4 and 688.4) is the most
abundant product. From these data it is obvious, that the
Assignment of the most intense peaks observed in the positive ion MALDI–TOF
mass spectra of PLPC oxidized by different oxidizing reagents.
m/z
Assignment
496.3 LPC 16:0 (+H+)
518.3 LPC 16:0 (+Na+)
579.4 Unidentified
636.4 1-palmitoyl-2-(8-oxo-octanoic acid)-sn-phosphatidylcholine (+H+)
638.4 1-palmitoyl-2-(8-hydroxyoctanoic acid)-sn-phosphatidylcholine (+H+)
650.4 1-palmitoyl-2-(9-oxo-nonaoic acid)-sn-phosphatidylcholine (+H+)
660.4 1-palmitoyl-2-(8-hydroxyoctanoic acid)-sn-phosphatidylcholine
(+Na+)
666.4 1-palmitoyl-2-(nonadioic acid)-sn-phosphatidylcholine (+H+)
672.4 1-palmitoyl-2-(9-oxo-nonaoic acid)-sn-phosphatidylcholine (+Na+)
688.4 1-palmitoyl-2-(nonadioic acid)-sn-phosphatidylcholine (+Na+)
720.6 Unidentified
722.6 Loss of headgroup from m/z 780.6
758.6 PC 16:0 18:2 (+H+) (PLPC)
780.6 PC 16:0 18:2 (+Na+) (PLPC)
788.6 PLPC + O2 ꢁ 2H (+H+)
790.6 PLPC + O2 (+H+)
804.6 PLPC + 2 O2 ꢁ H2O (+H+)
806.6 PLPC + 3/2 O2 (+H+)
810.6 PLPC + HOCl (+H+)
812.6 PLPC + O2 (+Na+)
814.6 Unidentified
822.6 PLPC + 2 O2 (+H+)
826.6 PLPC + 2HOCl ꢁ HCl (+H+)
862.6 PLPC + 2HOCl (+H+)
884.6 PLPC + 2HOCl (+Na+)
elements into an unsaturated fatty acyl residue upon oxidation
leads to a reduction of the strength of the ester linkage resulting in
an increased hydrolysis rate.
Additionally, the cleavage at one double bond position is
obvious by the generation of the characteristic aldehyde (1-
palmitoyl-2-(90-oxo-nonanoyl)-sn-glycero-3-phosphocholine) at
m/z = 650.4 (Itabe et al., 1996) and the corresponding carboxylic
acid (1-palmitoyl-2-(1,9-nonanedicarboxylic acid)-sn-glycero-3-
phosphocholine) at m/z = 666.4 (and to a much smaller extent at m/
z = 688.4 because the carboxylic acid undergoes exchange reactions
with alkali metal ions) (Podrez et al., 2002). Products with higher
masses in comparison to the original PLPC are not detectable under
these reaction conditions. Therefore, air-oxidation is the method of
choice when only chain-shortened products are of interest. In
contrast, such products are detectable (in addition to the products
incubation time has
a considerable effect: using prolonged
incubation times, only LPC 16:0, the carboxylic acid and the
758.6
780.6
(A)
758.6
788.6
650.4
496.3
666.4
(B)
666.4
826.6
826.6
758.6
496.3
862.6
790.6
688.4
666.4
688.4
(C)
496.3
652.4
(D)
500
550
600
650
700
m/z [Th]
750
800
850
Fig. 2. Selected positive ion MALDI–TOF mass spectra of PLPC oxidized by the Fenton reagent (FeCl2 and H2O2) in dependence on the incubation time. All spectra were
recorded with 9-AA as matrix and all peaks are marked by the corresponding m/z values. In (A) the spectrum of non-oxidized PLPC (control) is shown. PLPC was oxidized with
FeCl2 and H2O2 for 1 h (B), 2 h (C) and 16 h (D). All spectra are scaled in the way that the most intense peak possesses the same intensity. For details see text.

34
K. Zschörnig, J. Schiller / Chemistry and Physics of Lipids 184 (2014) 30–37
bis-chlorohydrin subsequent to HCl elimination (m/z = 826.6) are
detectable. In contrast, shorter incubation times favor the
generation of the corresponding aldehyde (m/z = 650.4). This is
not surprising because the carboxylic acid results from the
oxidation of the aldehyde.
pay very much attention to this problem since fraction (3B)
represents a mixture and our aim was to obtain chromatographi-
cally pure compounds.
Due to the identical lengths of their acyl residues it is not
surprising that these oxidation products (3B) cannot be differentiat-
ed by one-dimensional (normal phase) TLC. In contrast,
chain-shortened oxidation products can be easily differentiated:
in spot (3C) the aldehyde and in (3E) the corresponding carboxylic
acid can be easily identified although the molecular structure of the
compound with m/z = 814.6 (3C) remains to be elucidated. We do
also not yet have a convincing explanation of the peaks at
m/z = 638.4 and 660.4 detected in spot (3D) although it seems
likely that this compound has a very similar chemical structure as
the compound observed in trace (3C). It is well known that the
oxidation of polyunsaturated lipids may lead to the shift of a double
bondwhich wouldlead toamass differenceof 14 u incomparison to
the so far discussed products. For instance, Reis et al. (2005)
detected upon oxidation of PLPC in addition to the aldehyde at
m/z = 650.4anadditionalproductatm/z = 636.4whichwas assigned
to the chain-shortened aldehyde 1-palmitoyl-2-(8-oxo-octanoic
acid)-sn-phosphatidylcholine (one CH2 group results in a mass
difference of 14 u). However, the mass difference observed here is
only 12 u, not 14 u. A mass difference of 2 u might be explained by
the reduction of the aldehyde to the corresponding alcohol. Of
course, a reduction is not very likely because the reaction was
performed under strongly oxidizing conditions. Nevertheless, a
disproportionation of the aldehyde into the carboxylic acid and the
alcohol cannot be completely ruled out. As the quality of the
achieved MS/MS spectra was rather poor and did not allow a clear
assignmentoftheresidueinsn-2position(datanotshown),wehave
chosen an alternative way to confirm the molecular structure of the
product detected at m/z 638.4 and 660.4.
The enzyme phospholipase A2 is known to cleave exclusively
the fatty acyl residue in the sn-2 position of a given PL under
generation of the corresponding LPC and the related free fatty acid.
If the alcohol (8-hydroxyoctanoic acid) instead of linoleic acid
would be released, this should result in an altered polarity. Since
8-hydroxyoctanoic acid is commercially available from Aldrich,
this compound can be used to compare the related TLC migration
properties and, thus, to verify the potential presence of
8-hydroxyoctanoic acid. The corresponding developed TLC plate
is shown in Fig. 4. In lane (4C) the result of a partial digestion of
PLPC is shown as control. It is obvious that there are – in addition to
PLPC – only two products: LPC 16:0 and free linoleic acid.
Unfortunately, the digestion of the compounds present in the
fraction which results in the MS peaks at m/z 638.4 and
The so far performed experiments were done on an analytical
scale to optimize the experimental conditions to generate the
desired products in maximum yields. The oxidation of PLPC by
FeCl2 and H2O2 for 16 h was consequently chosen to isolate the
individual oxidation products on a larger scale by means of
preparative TLC. TLC is a highly established method for lipid
separation of even complicated mixtures (Fuchs et al., 2007b)
and has the most significant advantage that it can be easily
established in all laboratories because no sophisticated and/or
expensive equipment is necessary. It is also obvious from the
chromatogram (Fig. 3, left) that a one-dimensional TLC run is
already sufficient to separate the majority of the different
oxidation products of PLPC: even very similar compounds (e.g.,
the aldehyde and the carboxylic acid) can be separated and
subsequently isolated at a preparative scale. Of course, normal
phase TLC is normally more useful to separate different PL classes,
while reversed phase (RP) TLC (for instance C18 modified silica) is
the method of choice to separate lipids with different fatty acyl
residues. Unfortunately, RP TLC cannot be used at the so far
established conditions: using the dye primuline the entire RP TLC
plate is stained since the primuline binds to the apolar (C18)
residues (data not shown). This is the reason why we have used
exclusively non-modified silica.
The positive ion MALDI–TOF mass spectra of the identified six
TLC spots (subsequent to re-elution of the oxidized PLs from the
silica gel) are shown at the right hand of Fig. 3. Since the intensity
of the LPC on the TLC is very low, this part of the TLC plate is not
shown. The most apolar compound in spot (3A) possesses only low
intensity and can be unequivocally assigned to non-oxidized PLPC
with m/z = 758.6 and 780.6. The second spot (2B) is much more
intense and corresponds to a mixture of different compounds that
could not be resolved by 1D TLC: beside the peroxide (m/z = 790.6)
there is also a significant contribution of the bis-chlorohydrin (m/
z = 862.6) and an additional product corresponding to the loss of
HCl (m/z = 826.6) from the chlorohydrin. The last detectable
product at m/z = 804.6 is tentatively assigned to the addition of
two O2 molecules to PLPC followed by the loss of water. Due to the
very low concentration of this compound, we unfortunately failed
to record reasonable MS/MS spectra (even if an electrospray
ionization (ESI) MS with an ion trap mass analyzer is used) which
would make peak assignments unambiguous. However, we did not
Fig. 3. Left: image of a typical HPTLC plate used to separate the oxidation products of PLPC after incubation with the Fenton reagents (cf. Section 2) for 16 h. Staining was
performed by using the non-destructive dye primuline. Right: positive ion MALDI–TOF mass spectra recorded to confirm the identities of the lipids detectable in the
individual TLC fractions. Peak assignments are available in Table 1.

K. Zschörnig, J. Schiller / Chemistry and Physics of Lipids 184 (2014) 30–37
35
Table 2
Quantitative determination of the oxidation products of PLPC by means of the
Stewart assay. Because of the very low concentration of LPC, this compound cannot
be determined by the Stewart assay and is, thus, not listed in this table. Please note
that it was not possible to absolutely differentiate spots (c) and (d). Therefore, both
fractions were combined. For details see text.
Observed spot
Absorption
Amount (
m
g)
Yield (%)
(A)
(B)
(C) and (D)
(E)
Total
0.27
0.93
0.57
0.99
265
916
560
973
4.2
15.2
9.3
16.2
44.9
2714
Teuber et al. (2010)) was used to determine the quantitative
amounts of the individual oxidation products. As the outcome of
this assay is primarily determined by the headgroup of the PL of
interest (Stewart, 1980) and the phosphorylcholine headgroup is
not altered upon the used oxidation conditions, quantitative data
can be easily obtained. These data (which were obtained
subsequent to re-elution of the identified lipid spots from the
TLC plate (cf. Fig. 3)) are summarized in Table 2.
Fig. 4. TLC chromatogram to verify the generation of 1-palmitoyl-2-(8-hydro-
xyoctanoic acid)-sn-phosphatidylcholine as oxidation product of PLPC. In lane (C)
the products obtained by PLA2 digestion of PLPC (LPC and free linoleic acid) are
shown as control, while the digestion products of putative 1-palmitoyl-2-(8-
hydroxyoctanoic acid)-sn-phosphatidylcholine are shown in (B). The aqueous
reaction mixture was directly used in this case to avoid potential lipid losses upon
the extraction. This might be the reason why there are some pale spots that could
not be identified; these are presumably polar compounds that would be lost upon
extraction. Finally, isolated (commercially available) 8-hydroxyoctanoic acid is
shown in lane (A).
Although clear yield differences of the individual oxidation
products are obvious, it is also evident that there are significant
lipid losses, i.e., a certain portion of the lipid sticks tightly to the
silica gel and can be hardly extracted – even if a considerable
excess of solvents is used. However, this lipid loss only slightly
depends on the lipid structure (Teuber et al. (2010)): while there is
a significant loss of the absolute lipid amount, the relative moieties
of the individual PLPC oxidations products are presumably not
significantly altered. Using preparative TLC about 45% of the
originally applied PL can be recovered. A more detailed evaluation
whether the most pronounced lipid loss occurs during the
extraction from the aqueous phase (i.e., prior to the TLC) or as
the result of the TLC separation and the subsequent re-elution was
beyond the scope of this paper. Nevertheless, according to the data
shown in Table 2, the carboxylic acid and the aldehyde are the by
far most abundant products and can be isolated in amounts close to
1 mg. Therefore, it is concluded that the suggested method is
simple and convenient and allows even non-experts to prepare
oxidized, chain-shortened lipids when they are required in the
laboratory. The simple approach described in this work might be
particularly useful for cell biologists who would like to treat
dedicated cell lines with these lipid oxidation products.
However, it must be admitted that the lipid fractions (with the
exception of the aldehyde and the corresponding carboxylic acid)
isolated by our TLC approach are not pure. There are basically three
different approaches how this problem could be overcome: (a) by
using HPLC (b) applying reversed phase TLC and (c) by performing
two-dimensional TLC. We did not want to use HPLC because the
method should be as simple as possible and suitable HPLC
protocols are presumably not available in all laboratories. As
already discussed (vide supra) reversed phase TLC cannot be
combined with primuline staining and, thus, major modifications
of the workflow would be necessary. Therefore, we relied on
(c) and used 2D TLC whereby the very best results were obtained
when (commercially available) diol-modified silica was
used – although even these data are not very convincing. The
separation of Fenton-oxidized PLPC by 2D TLC is shown at the left
of Fig. 5 and the corresponding positive ion MALDI–TOF mass
spectra are shown at the right. It is obvious that exclusively spots
(B) and (C) represent chromatographically pure compounds while
all other spots (particularly (A)) represent still mixtures (cf. Table 1
for detailed peak assignments). This is of course disappointing
although we tried different stationary phases as well as solvent
systems. Therefore, it must be concluded that the so far performed
660.4 provides less convincing results (lane (4B)). This is caused by
two reasons: on the one hand, the concentration of this compound
is much smaller and, thus, the obtained bands of the chromato-
gram are comparably pale. On the other hand, an aqueous sample
without previous extraction was used in lane (4B). This was done to
minimize the potential loss of 8-hydroxyoctanoic acid by the
extraction with organic solvents. The use of the aqueous layer may
be one reason why there are several spots (presumably corre-
sponding to water-soluble, i.e., polar compounds) detectable
which are normally not visible if the organic extracts are
investigated. Nevertheless, there is a weak spot which nicely
corresponds to the migration properties of the pure 8-hydrox-
yoctanoic acid that was applied as reference in lane (4A). These
results are in agreement with the generation of 1-palmitoyl-2-(8-
hydroxyoctanoic acid)-sn-phosphatidylcholine upon the oxidation
of PLPC.
It is remarkable that 1-palmitoyl-2-(8-hydroxyoctanoic acid)-
sn-phosphatidylcholine is only detectable when the PLPC oxida-
tion is scaled up but is not detectable if smaller amounts of PLPC
oxidized. It should also be noted that the tendencies of Na+ adduct
generation are significantly different: while the products detected
in traces (3C) and (3D) have similar affinities to H+ and Na+, all
other fractions are dominated by the H+ adducts. Although this is
an interesting aspect from the mass spectrometric viewpoint (all
fractions should have similar alkali metal ion concentrations since
they were re-extracted from the same TLC plate), a more detailed
evaluation of the related mechanisms was beyond the scope of this
paper. It should be noted that this oxidation is also accompanied by
LPC generation. However, the LPC spot (as well as the correspond-
ing mass spectrum) is not shown in order to emphasize the other
spots. Thus, the compositions of the obtained fractions agree with
the data illustrated in Figs. 1 and 2.
Although MALDI MS is a convenient method to qualitatively
detect small amounts of lipids, it is not the method of choice to
obtain quantitative data. This particularly applies because the
different oxidation products may result in different ion yields
making the use of internal standards (which are not readily
available so far) mandatory. Therefore, the photometric approach
according to Stewart (that has also been successfully applied by

36
K. Zschörnig, J. Schiller / Chemistry and Physics of Lipids 184 (2014) 30–37
Fig. 5. Left: two-dimensional HPTLC separation of the oxidation products of PLPC after incubation with the Fenton reagents (cf. Section 2) for 16 h. Diol-modified silica was
used as stationary phase. Chloroform, methanol, water, ethanol, triethylamine (6.5/2/1/3.5/3.5, v/v/v/v/v) was used in the first dimension and chloroform, methanol, acetic
acid, water (90/40/12/2, v/v/v/v) in the second dimension. Right: positive ion MALDI–TOF mass spectra recorded from the positions indicated in the TLC image. Peak
assignments are available in Table 1.
TLC separations are only useful to isolate chain-shortened
products. Nevertheless, we are confident that even this problem
can be overcome in the future.
Fuchs, B., Schiller, J., Süß, R., Schürenberg, M., Suckau, D., 2007b. A direct and simple
methodofcouplingmatrix-assistedlaserdesorptionand ionization time-of-flight
mass spectrometry (MALDI–TOF MS) to thin-layer chromatography (TLC) for the
analysis of phospholipids from egg yolk. Anal. Bioanal. Chem. 389, 827–834.
Fuchs, B., Schiller, J., Süß, R., Zscharnack, M., Bader, A., Müller, P., Schürenberg, M.,
Becker, M., Suckau, D., 2008. Analysis of stem cell lipids by offline HPTLC-
MALDI–TOF MS. Anal. Bioanal. Chem. 392, 849–860.
Conflict of interest
Fuchs, B., Bischoff, A., Süss, R., Teuber, K., Schürenberg, M., Suckau, D., Schiller, J.,
2009. Phosphatidylcholines and -ethanolamines can be easily mistaken in
phospholipid mixtures: a negative ion MALDI–TOF MS study with 9-amino-
acridine as matrix and egg yolk as selected example. Anal. Bioanal. Chem. 395,
2479–2487.
The authors declare that there is no conflict of interest.
Transparency document
Fuchs, B., Süß, R., Schiller, J., 2010. An update of MALDI–TOF mass spectrometry in
lipid research. Prog. Lipid Res. 49, 450–475.
Fuchs, B., Bresler, K., Schiller, J., 2011. Oxidative changes of lipids monitored by
The Transparency document associated with this article can be
found in the online version.
MALDI MS. Chem. Phys. Lipids 164, 782–795.
Henry, S.A., Jiranek, V., Graves, J.A., 1998. Pleiotropic effects of the opi1 regulatory
mutation of yeast: its effects on growth and on phospholipid and inositol
metabolism. Microbiology 144, 2739–2748.
Acknowledgment
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