Isoketals form cytotoxic phosphatidylethanolamine adducts in cells

Article abstract of DOI:10.1194/jlr.M001040

Levuglandins and their stereo- and regio-isomers (termed isolevuglandins or isoketals) are γ-ketoaldehydes (IsoK) that rapidly react with lysines to form stable protein adducts. IsoK protein adduct levels increase in several pathological conditions including cardiovascular disease. IsoKs can induce ion channel dysfunction and cell death, potentially by adducting to cellular proteins. However, IsoKs also adduct to phosphatidylethanolamine (PE) in vitro, and whether PE adducts form in cells or contribute to the effects of IsoKs is unknown. When radiolabeled IsoK was added to HEK293 cells, 40% of the radiolabel extracted into the chloroform lower phase suggesting the possible formation of PE adducts. We therefore developed methods to measure IsoK-PE adducts in cells. IsoK-PE was quantified by LC/MS/MS after hydrolysis to IsoK-ethanolamine by Streptomyces chromofuscus phospholipase D. In HEK293 and human umbilical vein endothelial cells (HUVEC), IsoK dose-dependently increased PE adduct concentrations to a greater extent than protein adduct. To test the biological significance of IsoK-PE formation, we treated HUVEC with IsoK-PE. IsoK-PE dose dependently induced cytotoxicity (LC₅₀ 2.2 μM). These results indicate that cellular PE is a significant target of IsoKs, and that formation of PE adducts may mediate some of the biological effects of IsoKs relevant to disease. Copyright

Full text of DOI:10.1194/jlr.M001040

Isoketals form cytotoxic phosphatidylethanolamine
adducts in cells
C. Blake Sullivan, Elena Matafonova, L. Jackson Roberts II, Venkataraman Amarnath,
and Sean S. Davies1
Division of Clinical Pharmacology, Department of Pharmacology, and Department of Pathology, Vanderbilt
University, Nashville, TN
Abstract Levuglandins and their stereo- and regio-isomers
(termed isolevuglandins or isoketals) are ␥-ketoaldehydes
(IsoK) that rapidly react with lysines to form stable protein
adducts. IsoK protein adduct levels increase in several path-
ological conditions including cardiovascular disease. IsoKs
can induce ion channel dysfunction and cell death, poten-
tially by adducting to cellular proteins. However, IsoKs also
adduct to phosphatidylethanolamine (PE) in vitro, and
whether PE adducts form in cells or contribute to the ef-
fects of IsoKs is unknown. When radiolabeled IsoK was
added to HEK293 cells, 40% of the radiolabel extracted
into the chloroform lower phase suggesting the possible
formation of PE adducts. We therefore developed methods
to measure IsoK-PE adducts in cells. IsoK-PE was quantified
by LC/MS/MS after hydrolysis to IsoK-ethanolamine by
Streptomyces chromofuscus phospholipase D. In HEK293 and
human umbilical vein endothelial cells (HUVEC), IsoK
dose-dependently increased PE adduct concentrations to a
greater extent than protein adduct. To test the biological
significance of IsoK-PE formation, we treated HUVEC with
IsoK-PE. IsoK-PE dose dependently induced cytotoxicity
(LC50 2.2 ␮M). These results indicate that cellular PE is a
significant target of IsoKs, and that formation of PE adducts
may mediate some of the biological effects of IsoKs relevant
to disease.—Sullivan, C. B., E. Matafonova, L. J. Roberts II,
V. Amarnath, and S. S. Davies. Isoketals form cytotoxic
phosphatidylethanolamine adducts in cells. J. Lipid Res.
2010. 51: 999–1009.
drew immediate interest was their extreme reactivity to-
ward the lysyl residues of proteins, with reaction rates sev-
eral orders of magnitude faster than the ␣, ␤-unsaturated
aldehyde 4-hydroxynonenal, perhaps the most widely stud-
ied lipid peroxidation product (5, 6). This increased reac-
tivity derives from the facile formation of stable pyrrole
adducts on primary amines. In the presence of oxygen,
these pyrrole adducts convert over time into highly stable
lactam adducts, as well as hydroxylactam adducts (5, 7).
Increased concentrations of IsoK protein adducts have
been observed in vivo during a wide variety of conditions
associated with inflammation and oxidative stress, includ-
ing atherosclerosis, end stage kidney disease, myocardial
infarction, Alzheimer’s Disease, glaucoma, hyperoxia, al-
lergic inflammation, and experimental sepsis (8–14).
Whether IsoK adducts contribute to pathogenesis of
these diseases or are simply byproducts of the disease re-
mains an active area of investigation. Evidence supporting
a potential role in pathogenesis comes from treatment of
cultured cells with exogenous IsoK, which induces a vari-
ety of responses relevant to disease, including increased
macrophage uptake of LDL, activation of platelet aggrega-
tion via p38-MAP kinase, inhibition of sodium and potas-
sium channels, inhibition of proteasome function, and
cytotoxicity (9, 15–19).
The mechanism by which IsoK induces these effects has
been presumed to be its adduction to cellular proteins,
because of the high concentration of lysyl residues in cells.
However, these lysyl residues are not the only potential tar-
get of IsoK present in cells. In vitro, IsoK can form adducts
with a variety of other primary amines including the amino-
phospholipid phosphatidylethanolamine (PE) (20–22).
In phosphate buffer, the reaction rate of ␥-ketoaldehydes
to form pyrrole adducts with ethanolamine, the head
group of the PE, is 4.4-fold faster than with lysine (20).
Supplementary key words cytotoxicity • endothelial cell • inflamma-
tion • isoketal • levuglandin • oxidative stress • phosphatidyletha-
nolamine • phospholipase D
Levuglandins and their regio- and stereo-isomers (isole-
vuglandinsorisoketals)arehighlyreactive␥-ketoaldehydes
(IsoK) formed by nonenzymatic rearrangement of prosta-
glandin H2 and their free radical generated counterparts
(H2-isoprostanes) (1–5) (Fig. 1). One feature of IsoKs that
This work was supported by Vanderbilt junior faculty start-up funds and the
National Institutes of Health Grants HL-079365 and GM-42056. Its contents
are solely the responsibility of the authors and do not necessarily represent the
official views of the National Institutes of Health or other granting agencies.
Abbreviations: CID, collision induced disassociation; HEK293, hu-
man embryonic kidney; HUVEC, human umbilical vein endothelial
cell; IsoK, ␥-ketoaldehyde isomer; MRM, multiple reaction monitoring;
PE, phosphatidylethanolamine; PLD, phospholipase D.
1 To whom correspondence should be addressed.
Manuscript received 11 August 2009 and in revised form 24 November 2009.
Published, JLR Papers in Press, November 24, 2009
DOI 10.1194/jlr.M001040
e-mail: sean.davies@vanderbilt.edu
Copyright © 2010 by the American Society for Biochemistry and Molecular Biology, Inc.
This article is available online at http://www.jlr.org
Journal of Lipid Research Volume 51, 2010
999

Fig. 1. Pathways leading to the formation of ␥-ketoaldehyde isomers and adducts. In vivo, arachidonic acid
is converted to bicyclic endoperoxides either enzymatically by the action of cyclooxygenase working on free
arachidonic acid (forming prostaglandin H2) or nonenzymatically by free radical mediated lipid peroxida-
tion of esterified arachidonic acid (forming H2-isoprostanes). Nonenzymatic rearrangement of these bicyclic
endoperoxide generates IsoKs, termed levuglandins and isolevuglandins (alternatively named isoketals).
IsoKs react with the lysyl residues of proteins, and potentially other primary amines including the etha-
nolamine head group of PE, to rapidly form pyrrole adducts which convert to lactam and hydroxylactam
adducts in the presence of oxygen. IsoK, ␥-ketoaldehyde isomer; PE, phosphatidylethanolamine.
Oxidation of multilaminar vesicles containing PE or of iso-
lated high density lipoprotein in vitro produces pyrrolized
phospholipids (measured by reactivity with Ehrlich re-
agent), suggesting the robust modification of PE by pyr-
role forming lipoxidation products under these conditions
(23).
Although the biological effects of IsoK-modified PE
have not been studied, modification of the ethanolamine
headgroup of PE by IsoK is likely to dramatically affect the
physical properties of PE as it converts the neutral PE to an
acidic phospholipid and significantly increases headgroup
bulk. PE modified by IsoK might also activate receptor-
mediated signaling in a manner analogous to N-acyl-PE,
which suppresses food intake via cFOS expression in neu-
ropeptide Y neurons, and suppresses phagocytosis via Rac1
and Cdc42 in macrophages (24, 25). We therefore sought
to determine the extent to which IsoK adducts to PE com-
pared with proteins and whether IsoK-PE could mediate
IsoK-induced cytotoxicity.
MATERIALS AND METHODS
Materials
L-␣-phosphatidylethanolamine (PE), transphosphatidylated
from chicken egg phosphatidylcholine was purchased from
Avanti Polar lipids (Alabaster, AL). According to the manufac-
turer and our LC/MS analysis, the major species of PE in this
product is 1-palmitoyl-2-oleolyl-sn-glycerophosphotidyletha-
nolamine (C34:1 PE). All solvents were HPLC grade and were
purchased from EMD Biosciences. Streptomyces chromofuscus phos-
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Journal of Lipid Research Volume 51, 2010

pholipase D (PLD) was purchased from Enzo Life Sciences
International (Plymouth Meeting, PA), and Proteinase K was
purchased from Clontech (Mountain View, CA). Human embry-
onic kidney (HEK293) cells were purchased from ATCC
(Manassas, VA). Endothelial cells derived from donated human
umbilical cords (HUVEC) were a kind gift from Dr. Matthew
Duvernay. Endothelial cell basal medium was purchased from
Lonza Walkersville Inc. (Walkersville, MD). [2H4]ethanolamine
was purchased from Cambridge Isotopes (Cambridge, MA). All
other chemicals were from Sigma-Aldrich.
[2H4]ethanolamine. Triethylammonium acetate (1M) was used
in some reactions in place of PBS. IsoK-Etn was then extracted
with 3 vols chloroform/methanol (2:1), and the resulting product
analyzed by mass spectrometry. For quantification of resulting
pyrrole product, the reaction mixture was treated with Ehrlich
reagent and the absorbance at 580 nm measure by spectrometer.
Hydrolysis of IsoK-PE by PLD
Twenty-five nmol each of IsoK-PE reaction mixture was added
to microfuge tubes, along with 784 pmol of IsoK-[2H4]Etn inter-
nal standard and dried under nitrogen. Additional samples con-
taining internal standard only were also prepared. The lipids
were resuspended in 50 ␮l methanol, vortexed, 450 ␮l HBSS
added, and then sonicated for 5 min. 10 ␮l of PLD (>50,000 U/
ml) was added to the appropriate tubes and all samples were in-
cubated overnight at 37°C. Then 1.5 ml of chloroform/methanol
(2:1) was added to extract IsoK-Etn, the chloroform layer dried
down under nitrogen, resuspended in 50 ␮l methanol, and 10 ␮l
injected unto LC/MS/MS for analysis.
Synthesis of 15-E2-isoketal and [3H4]15-E2-isoketal methyl
ester
15-E2-isoketal (IsoK) was prepared from dimethoxyacetal pre-
cursor (DMA) in methylene chloride as previously described
(26) and a stock of 10 mM IsoK prepared in DMSO by evapora-
tion of methylene chloride under nitrogen. For synthesis of
[3H4]15-E2-IsoK methyl ester, oct-1-yn-3-ol in acetone was oxi-
dized with chromic acid (room temperature, 1 h) to oct-1-yn-3-
one. The ketone was purified by distillation (20–25°C/1 Torr)
and reduced with NaB3H4 to [3-3H4]oct-1-yn-3-ol. The latter was
then incorporated into IsoK methyl ester as described (26).
Mass spectrometry analysis
For limited mass scans of the products of the reaction between
IsoK and the PE preparation, samples were injected into the
ThermoFinnigan Quantum electrospray ionization triple quad-
rapole mass spectrometer operating in negative ion mode and
scanning between m/z 600 and 1200. For collision induced disas-
sociation (CID) spectrum, product ion scanning was performed
on the m/z 1032.7 precursor ion, using argon gas in the collision
cell. Collision energy was set at 50 eV. Similar analysis was per-
formed on the PE preparation alone, with scanning performed at
m/z 600 to m/z 1200, and a CID spectrum obtained for the pre-
cursor ion at m/z 716.5.
Treatment and fractionation of HEK293 cells
HEK293 cells were grown on 100mm dishes to >90% conflu-
ence. Cells were then scraped, transferred, washed twice in
Dulbecco Phosphate Buffer Saline (DBPS), and then resus-
pended in 2 ml DPBS. [3H4]15-E2-IsoK methyl ester in DMSO
(final concentration 1 ␮M) was added to the resuspended cells
and incubated for 2 h while gently rocking. To separate protein
adducts from phospholipid adducts, 10 ml of chloroform/meth-
anol (2:1) was added, and the lower (chloroform) phase removed
for counting by liquid scintillation. The protein pellet at the
interface between the aqueous and chloroform phase was then
also removed and counted. To measure the amount of DNA
adducts, replicate HEK293 cells were treated with [3H4]15-E2-
IsoK methyl ester, and after the 2 h incubation, the cells were
pelleted by centrifugation and resuspended in 0.5 ml lysis buffer
(100 mM Tris HCl, pH 8.5, 5 mM EDTA, 0.2% SDS, 200 mM
NaCl, 100 ug Proteinase K/ml). The resulting lysate was precipi-
tated with isopropanol, and the supernatant discarded. The DNA
precipitant was then counted by liquid scintillation.
For LC/MS/MS analysis of IsoK-PE, gradient HPLC was per-
formed using a Agilent Zorbax XDB-C8 2.1 × 50 mm 5 ␮m col-
umn at a flow rate of 250 ␮l/min starting at 99% Solvent A
(methanol/acetonitrile/1 mM ammonium acetate (60:20:20))
for 1 min, then a gradient ramp to 99% Solvent B (1 mM ammo-
nium acetate in ethanol) at 7 min, and then holding at 99% Sol-
vent B for 1 additional min before returning to the starting
conditions. The HPLC eluant was directly connected to the mass
spectrometer operating in multiple reaction monitoring (MRM)
mode for m/z 1032.7→255.1@50eV and 1032.7→255.1@50eV for
IsoK-C34:1 PE and m/z 716.6→255.1@50eV for C34:1 PE.
For limited mass scans of the products of the reaction between
IsoK and Etn or IsoK and [2H4]Etn, samples were injected into
the mass spectrometer operating in positive ion mode and scan-
ning between m/z 350 and 430. For CID spectrum, product ion
scanning was performed on the m/z 378.3, 382.3, 394.3, 398.3,
410.3, and 414.3 ions using argon gas in the collision cell. Colli-
sion energy was set at 25 eV. For LC/MS/MS analysis of IsoK-Etn,
gradient HPLC was performed using a XDB-C8 column at a flow
rate of 500 ␮l/min starting at 100% Solvent A (water with 0.1%
acetic acid) with a gradient ramp to 100% Solvent B (metha-
nol with 0.1% acetic acid) at 3 min, and then holding at 100%
Solvent B for 1 additional min before returning to the starting
conditions. The HPLC eluant was directly connected to the
mass spectrometer operating in multiple reaction monitoring
mode for m/z 378.3→152.2@-25eV (IsoK-Etn pyrrole), m/z
394.3→178.2@-25eV (IsoK-Etn lactam), m/z 382.3→156.1@-25eV
(IsoK-[2H4]Etn pyrrole), and m/z 398.3→182.2@-25eV (IsoK-
[2H4]Etn lactam).
Synthesis of modified PE and ethanolamine
Synthesis of IsoK-PE was performed by two methods. IsoK-PE
was initially synthesized as described previously (21). In brief,
1.25 ␮mol PE in CHCl3 was dried down under nitrogen and re-
suspended in 1ml of a two phase mixture of ether/PBS (4:1). 125
nmol IsoK (0.1 molar equivalents) in 12.5 ␮l DMSO was added to
the sample and incubated for 2 h at 37°C. After incubation, the
ether layer was dried off under nitrogen and 1ml chloroform/
methanol (2:1) added. The resulting chloroform (lower) phase
was transferred, dried under nitrogen, resuspended in 0.5 ml
methanol/chloroform (9:1) and stored at Ϫ70°C until use. Sub-
sequent experiment used PE in a single phase mixture of 1 M
triethylammonium acetate/chloroform/ethanol (1:1:3) in place
of the two phase ether/PBS solvent. IsoK-PE synthesized in this
manner was used without further purification. The reaction of
DMA with PE was performed with PBS/ether in a similar manner
as the reaction of IsoK with PE, using 125 nmol DMA (0.1 molar
equivalents) in 12.5 ␮l DMSO instead of IsoK. For the vehicle
reaction, 12.5 ␮l DMSO was added to the PE.
Quantification of IsoK protein and IsoK-PE adducts in
IsoK treated HEK293 and HUVEC cells
IsoK (0–10 ␮M) was incubated with human embryonic kidney
To synthesize IsoK-Etn, 20 ␮l neat ethanolamine was added to
20 nmol IsoK in 200 ␮l PBS and incubated for 2 h at 37°C. IsoK-
[2H4]Etn was synthesized in a similar manner using 20 ␮l neat
(HEK293) plated in duplicate wells of 6 well dishes containing
Cytotoxic isoketal PE adducts
1001

evaporated under nitrogen and resuspended in HBSS containing
0.1% BSA (HBSS/A). To determine the concentration depen-
dence of IsoK-PE induced HUVEC cytotoxicity, the concentra-
tion of IsoK, DMA, or vehicle treated PE were calculated from the
amount of IsoK or DMA added to PE, respectively. Therefore, for
each stated treated PE concentration, the total PE concentration
(modified and unmodified PE) was equivalent for each type of
treatment and was exactly 10- fold higher than the stated treated
PE concentration. IsoK-, DMA-, or vehicle-treated PE (0.1–30 ␮M
final concentration) were each added to six replicate wells of
HUVEC. Untreated control wells were used to determine maxi-
mum viability. Cells were incubated with their respective treated
PE for 22 h, and then 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphe-
nyltetrazolium bromide (MTT) added to each well for an addi-
tional two h, the culture media replaced with isopropanol and
the extent of MTT conversion measured by the absorbance at
570 nm as previously described. Viability was normalized to the
HBSS/A only treated cells.
To determine the amount of IsoK-PE that entered the cells
during the incubation process to that amount of IsoK-PE formed
Fig. 2. Distribution of IsoK adducts in cells. [3H]IsoK methyl es-
ter (1 ␮M) was added to HEK293 cells for two h, and the distribu-
tion of the resulting adducts measured by liquid scintillation
counting of the [3H] extracted into lower chloroform organic
phase that includes phospholipids, into the protein interphase,
and into isolated DNA. IsoK, ␥-ketoaldehyde isomer.
HBSS for two h. The treatment media was then removed care-
fully to avoid disturbing the cell layer, and any unreacted IsoK in
the treatment media extracted with hexane and measured by
negative ion electrospray LC/MS/MS by monitoring MRM tran-
sitions m/z 351→m/z 271. To measure IsoK adducts in cells, 2 ml
HBSS was immediately added to the plated cells after removing
incubation media, the cell scraped, and cell and wash media
transferred to clean centrifuge tubes. Six ml of chloroform/
methanol (2:1) was added to each sample to extract the phos-
pholipids, and the lower chloroform layer containing phospho-
lipids was transferred to a new tube. The protein pellet at the
interphase was also transferred to a separate tube. After addition
of 1 nmol of IsoK-[2H4]Etn as the internal standard, the chloro-
form layer was dried down under nitrogen gas and resuspended
in 50 ␮l methanol, vortexed, and then 450 ␮l HBSS added. The
entire mixture was then sonicated for 5 min. Fifteen uL PLD was
added to the samples and incubated overnight at 37°C. The sam-
ples were extracted with 1.5 ml chloroform/methanol (2:1). The
chloroform (lower) layer was transferred, dried down under ni-
trogen, and resuspended in 50 ␮l methanol for LC/MS/MS anal-
ysis. Analysis of IsoK-protein adducts as IsoK-lysyl-lactam adduct
after protease digestion was performed on the protein pellet as
previously described after addition of 10 pmol IsoK-[13C615N2]lysyl-
lactam the internal standard.
Human umbilical vein endothelial cells (HUVEC; passage 6-8)
were plated in 6 well dishes coated with 0.2% gelatin and allowed
to grow to >90% confluence. The EBM medium was removed
and the cells washed three times with HBSS and then duplicate
wells treated with 0-1 ␮M IsoK. Analysis was then performed in
similar manner as for HEK293 cells, except that 2 ml 0.25%
Trypsin EDTA solution was added to plated cells, allowed to incu-
bate for 3 min, and the cells and wash media removed and
analyzed.
Fig. 3. IsoK reacts with PE to form pyrrole adduct. IsoK was incu-
bated with a commercial preparation of PE and negative ion lim-
ited mass scanning from m/z 600 to 1200 performed on the starting
reactants and reaction mixture to identify novel ions in the reac-
tion mixture. A: Limited mass scans of the commercial PE prepa-
ration containing primarily C34:1 PE (m/z 716.7) as well as some
C34:2 PE, C36:1 PE, and C36:2 PE. B: Limited mass scans of the
IsoK preparation. C: Limited mass scans of the reaction mixture
of PE and IsoK showing novel peaks with +316 amu mass shifts
from the PE preparation, consistent with the formation of
IsoK-PE pyrrole adducts. IsoK, ␥-ketoaldehyde isomer; PE,
phosphatidylethanolamine.
HUVEC cytotoxicity
HUVEC (passage 6-8) in EBM basal medium were plated into
96 well plates coated with 0.2% gelatin and allowed to grow to
>90% confluence. The EBM medium was removed and the cells
washed three times with HBSS. PE treated with 0.1 molar equiva-
lent IsoK, DMA, or vehicle was prepared as described above,
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Journal of Lipid Research Volume 51, 2010

when IsoK was directly incubated with cells, replicate plates of
HUVEC cells were treated with 3 ␮M IsoK-PE for 2 h, the incu-
bation media removed, and IsoK-PE measured after PLD incu-
bation as described for IsoK treated HUVEC cells.
that only 8% of the radiolabel associated with the pro-
tein interphase, while 40% of the radiolabel associated
with the chloroform lower phase that includes phospho-
lipids (Fig. 2). The remaining radiolabel associated with
the upper aqueous phase potentially containing adducts
with small peptides, polyamines, nucleic acids, and other
polar amines. When DNA was isolated directly from rep-
licate treated cells, 2% of the radiolabel was associated
with DNA. The distribution of radiolabel into the lower
chloroform layer is consistent with the formation of sub-
stantial amounts of adducts with aminophospholipids
such as PE; however, [3H4]IsoK methyl ester is itself
quite lipophilic, so that the radiolabel in the lower chlo-
roform layer could also represent unreacted IsoK or me-
tabolites. Therefore, we sought definitive evidence for
the formation of IsoK-aminophospholipid adducts in
treated cells.
RESULTS
Potential formation of nonprotein adducts in IsoK
treated cells
To identify significant cellular targets of IsoKs, a ra-
diolabeled methyl ester ([3H4]IsoK-methyl ester) of 15-
E2-IsoK, a representative IsoK, was incubated with
HEK293 cells and the resulting adduct species separated
by chloroform/methanol extraction. HEK293 cells were
chosen for initial cellular studies because of their rapid
growth characteristic and ease of maintenance. We found
Fig. 4. Development of IsoK-PE LC/MS/MS assay.
A: CID spectrum of m/z 1032.7 ion. B: Interpretation
of CID spectrum. C: MRM ion chromatographs from
LC/MS/MS analysis of IsoK-PE preparation for C34:1
PE and IsoK-34:1 PE pyrrole. IsoK, ␥-ketoaldehyde
isomer. CID, collision induced disassociation; IsoK,
␥-ketoaldehyde isomer; MRM, multiple reaction
monitoring; PE, phosphatidylethanolamine.
Cytotoxic isoketal PE adducts
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Characterization of the reaction of IsoK with
phosphatidylethanolamine
after overnight incubations, in contrast to our previous ob-
servations with IsoK protein adducts, probably because the
organic solvents used for the PE reaction tend to exclude
oxygen to a greater extent than aqueous buffers used for
protein reactions. However, when the final PE product was
dried down and left exposed to room air for several h, we
observed a peak at 1048.7, consistent with the formation of
a lactam adduct (not shown.) The CID spectrum of m/z
1032.7 includes major product ions at m/z 255, 281, 391,
512, and 750 (Fig. 4A). The product ions at m/z 255 and
m/z 281 are consistent with the two fatty acid side chains
(Fig. 4B), while the product at m/z 391 is consistent with a
dehydrated C16:0 lysoPA product. The product ion at m/z
512 is consistent with the neutral loss of the two acyl chains
from IsoK-PE, and the product ion at m/z 750 is consistent
with the neutral loss of the sn-2 acyl chain. Given the char-
acteristic fragmentation of IsoK-C34:1 PE, the expected
product ions formed by other diacyl PE modified by IsoK
can be readily predicted. Therefore, we used IsoK-C34:1
PE to develop an appropriate LC/MS/MS assay to mea-
sure the amount of IsoK-PE and PE. Modification of the
HPLC method previously described for the separation of
N-acyl-PE from PE, achieved significant separation of IsoK-
C34:1 PE from unmodified C34:1 PE (Fig. 4C).
The amount of PE adduct formed in cells should de-
pend both on the relative amount of PE and the reaction
rate of PE versus other primary amines. The model
␥-ketoaldehyde, 4-oxopentanal (OPA) reacts with etha-
nolamine 4.4 times faster than the reaction rate with
N-acetyl lysine (20). We determined the second order re-
action rate of OPA and PE using methods similar to our
previous study, except that we used a more lipophilic sol-
vent system (1 M triethylammonium acetate/chloroform/
ethanol 1:1:3 v/v/v) to maintain the solubility of the final
product. In this lipophilic system, the reaction rate of OPA
with PE was 4.5 × 10Ϫ3 MϪ1 sϪ1, which was 25 times faster
than the reaction rate of OPA with N-acetyl lysine (0.18 ×
10Ϫ3 MϪ1 sϪ1) that we had previously measured.
To characterize the IsoK-PE adduct and develop an
analytical method to quantify these adducts, we reacted
a commercial preparation of PE primarily containing
1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine
(C34:1 PE) and lesser amounts of C34:2 PE, C36:1 PE,
and C36:2 PE (Fig. 3A) with a representative IsoK, 15-E2-
IsoK (Fig. 3B) and compared the resulting products
(Fig. 3C) by negative ion mass spectrometry. Addition of
IsoK decreased the amount of signals at m/z 716.7
(C34:1PE), m/z 714.7 (C34:2 PE), m/z 744.7 (C36:1 PE),
and m/z 742.7 (C36:2 PE) and led to the formation of new
signals at m/z 1032.7, 1030.7, 1060.7, and 1058.7. The +316
amu mass shift of these novel peaks from the starting PE is
consistent with formation of IsoK-pyrrole adducts for
C34:1, C34:2, C36:1, and C36:2 PE, respectively. Under
our reaction conditions, we did not observe formation of
oxidized pyrrole adducts (lactam or hydroxylactam) even
We then treated HEK293 cells with vehicle or 10 ␮M
IsoK and monitored the formation of IsoK-PE using MRM
of transitions between the predicted precursor ion of vari-
ous IsoK-PE species containing C16:0 or C18:0 acyl chains
and their appropriate product ions at m/z 255 (C16:0) or
m/z 283 (C18:0). In HEK293 cells treated with vehicle (0
␮M IsoK), there were virtually no signals above the noise
when monitoring predicted IsoK-PE precursor ions (Fig. 5,
left panel). In contrast, addition of 10 ␮M IsoK to HEK293
Fig. 5. IsoK treatment of HEK293 cells forms IsoK-
PE. HEK293 were treated either with vehicle (0 ␮M
IsoK, left panel) or 10 ␮M IsoK (right panel) for 2 h.
MRM ion chromatographs of the IsoK pyrrole ad-
ducts for the most abundant diacyl PE species. IsoK,
␥-ketoaldehyde isomer; MRM, multiple reaction
monitoring; PE, phosphatidylethanolamine.
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Cytotoxic isoketal PE adducts
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resulted in significant signals at the appropriate retention
times for a number of potential IsoK-PE species (Fig. 5,
right panel). The rank order of peak height for these pre-
dicted IsoK-PE species were C34:1 (m/z 1032.7) > C36:1
(m/z 1060.7) > C38:4 (m/z 1082.7) > C34:0 (m/z 1034.7) >
C36:0 (m/z 1062.7) > C36:4 (m/z 1054.7). The average ra-
tios of the peak height for IsoK-C34:1 PE (m/z 1032.7→m/z
255) compared with C34:1 PE (m/z 716.7→m/z 255) in
cells treated with 10 ␮M IsoK was 0.013. Although poten-
tial differences in ionization efficiency between PE and
IsoK-PE preclude exact quantitation based on these peak
heights, these results would suggest that 10 ␮M IsoK po-
tentially modified roughly 1–2% of the C34:1 PE.
that the imine is an unstable intermediate that converts
relatively rapidly to the pyrrole adduct. The reaction of
IsoK with [2H4]Etn generated products at m/z 382, 398,
400, and 414, and the CID spectrum of these individual
products showed 4 amu mass shifts from the CID spectrum
of their nonisotopically labeled counterparts (not shown).
We then utilized these ions to develop a suitable stable iso-
tope dilution LC/MS/MS assay for IsoK-Etn. Under these
conditions, the hydroxylactam adduct eluted with a reten-
tion time of 2.9 min, the lactam product eluted with a re-
tention time of 3.0 min, and the pyrrole adduct with a
retention time of 3.1 min (Fig. 7). When the products of
the reaction with either IsoK only, Etn only, or IsoK with
Etn were monitored using this method, signal for the pyr-
role, lactam, and hydroxylactam adduct were only present
in the reaction containing both IsoK and Etn. Analysis of
the IsoK with Etn reaction initially showed that the pyrrole
product gave the most intense signal, but after 48 h, the
predominant signal was that of the lactam adduct. There-
fore, both pyrrole and lactam were chosen for monitoring
in subsequent experiments.
The phospholipase D from S. chromofuscus is able to ef-
ficiently hydrolyze PE modified with long chain N-acyl
groups, suggesting that this enzyme might be able to rec-
ognize IsoK-PE as well. We therefore determined whether
IsoK-PE could be converted to IsoK-Etn utilizing treatment
with this enzyme. IsoK-[2H4]Etn was added as an internal
standard to samples with PLD only, IsoK-PE only, or IsoK-
PE and PLD. Sample with S. chromofuscus PLD only did not
generate significant IsoK-Etn signal, (Fig. 8, left panel),
but gave strong signals for both the IsoK-[2H4]Etn internal
standard pyrrole and lactam adducts. The sample with
IsoK-PE only gave similar results (Fig. 8, middle panel.) In
contrast, the sample with S. chromofuscus PLD treated IsoK-
PE gave very strong signals with appropriate retention
times for both IsoK-Etn pyrrole and lactam adducts (Fig. 8,
right panel.) These results demonstrate that S. chromofus-
cus PLD converts IsoK-PE to IsoK-Etn. Although the origi-
nal IsoK-PE preparation contained very little lactam
adduct, the final PLD hydrolysis product contains signifi-
cant amounts of the lactam adduct, suggesting that during
the overnight incubation with buffer and lipase the pyr-
role adduct undergoes conversion to the lactam adduct.
IsoK-PE is a substrate for phosholipase D
Although individual PE species modified by IsoK in cells
can be measured by this method, relatively high concen-
trations of IsoK (10 ␮M) were required to detect signifi-
cant signals above noise so that this method would not be
very useful for cell based studies analyzing lower concen-
trations of IsoK. Furthermore, accurate quantitation of
the total IsoK-PE formed in cells after various stimuli would
require monitoring each of the individual diradyl IsoK-PE
species and summing their individual abundances. With
potentially more than 30 individual diradyl PE species
present in cells, accurate measurement of IsoK-PE by this
method appeared unwieldy. Therefore, we sought to de-
velop an analytical method that could quantify the entire
cellular content of IsoK-PE in a single analysis. One poten-
tial strategy to achieve this goal is to enzymatically or
chemically hydrolyze the modified ethanolamine (Etn)
head group and analyze the resulting IsoK-Etn products.
To determine if IsoK-Etn could be quantified by stable
isotope dilution LC/MS/MS, we reacted IsoK with an ex-
cess of Etn or [2H4]Etn and analyzed the resulting prod-
ucts by limited mass scan positive ion electrospray mass
spectrometry. The most abundant product formed by the
two h reaction of IsoK with Etn was a m/z 378 ion, consis-
tent with an IsoK-Etn pyrrole adduct (Fig. 6A). Additional
products at m/z 394, m/z 396, and m/z 410 are consistent
with formation of the expected lactam, imine, and hy-
droxylactam IsoK-Etn products. The CID spectrum of the
m/z 378 ion showed a major product ion at m/z 152,
along with ions with m/z 81, 138, 152, 180, 194, 222, 234,
251, 278, and 360 (Fig. 3B). The CID spectrum of the m/z
394 ion showed product ions at m/z 153, 166, 178, 192,
206, 250, 274, 358, and 376. The CID spectrum of the m/z
410 ion showed product ions at m/z 71, 99, 147, 167, 187,
215, 276, 313, and 374. The CID spectrum of the m/z 396
ion showed product ions at m/z 62, 221, 281, 291, 299,
317, 360, and 378 consistent with an imine adduct (not
shown), but this product was not studied further because
our experience with IsoK protein adducts suggested
PE is a significant target of IsoK in cells
We then utilized this methodology to quantify the total
amount of IsoK-PE formed in cells treated with IsoK and
compared this to the total amount of IsoK-protein adduct.
HEK293 cells were treated with IsoK (0–10 ␮M) for 2 h,
the treatment media removed and analyzed for unreacted
IsoK, and the levels of PE and protein adduct measured in
the cells. On average, less than half of the added IsoK
(47%) was still present in the incubation media after 2 h of
Fig. 6. Identification of IsoK ethanolamine (Etn) adducts. IsoK was incubated with Etn and resulting products analyzed by positive ion
electrospray ionization mass spectrometry. A: Limited mass scan spectrum of the reaction of IsoK with Etn produces ion consistent with
pyrrole (m/z 378), lactam (m/z 394), Schiff base (m/z 396), and hydroxylactam (m/z 410) adducts. B: CID spectrum and interpretation of
m/z 378 ion. C: CID spectrum and interpretation of m/z 394 ion. D: CID spectrum and interpretation of m/z 410 ion. CID, collision induced
disassociation; IsoK, ␥-ketoaldehyde isomer.
1006
Journal of Lipid Research Volume 51, 2010

dothelial cells treated with IsoK. Treatment of HUVEC
with IsoK (0-1 ␮M) also resulted in the formation of
IsoK-PE adducts and protein adducts (Fig. 9B), and again
the amount of PE adduct formed was greater than that of
protein adduct. These results indicate that PE is a signifi-
cant target of IsoK in cells, raising the possibility that
IsoK-PE might mediate some of the effects of IsoK on
cells.
IsoK-PE decreases the viability of endothelial cells
To test whether IsoK-PE could induce similar effects as
IsoK, we examined the ability to IsoK-PE to induce en-
dothelial cell death. Endothelial cell injury is a hallmark of
atherosclerosis and IsoKs are some of the most potent cy-
totoxins formed by lipid peroxidation. To test whether
IsoK-PE induced endothelial cytotoxicity, we prepared
IsoK-PE as before, along with PE treated with the DMSO
(vehicle) or with the inactive dimethoxy acetal isoketal
precursor (DMA). Human umbilical vein endothelial cells
were incubated overnight with 0.1–30 ␮M IsoK, DMA, or
vehicle treated PE and the resulting changes in cell viabil-
ity measured. Vehicle and DMA treated PE had no signifi-
cant effect on viability at any of the tested concentrations,
but IsoK-PE concentration dependently reduced the via-
bility of HUVEC (LC50 2.2 ␮M) (Fig. 10). To compare the
amount of IsoK-PE that actually entered the cells under
these conditions with the amount of IsoK-PE formed when
HUVEC where directly treated with IsoK, we incubated
HUVEC for 1 h with 3 ␮M IsoK-PE, removed the treatment
media from the cells, and then measured the IsoK-PE
adduct/ ␮g DNA in the HUVEC. We found that treatment
with 3 ␮M IsoK-PE resulted in 1.4 pmol adduct/␮g DNA
in the treated HUVEC, approximately half the amount of
IsoK-PE formed when adding 0.3 ␮M IsoK to the HUVEC.
These results are consistent with the notion that IsoK in-
duces cytotoxicity at least in part by reacting with PE to
form a cytotoxic product.
Fig. 7. LC/MS/MS analysis of IsoK-Etn adducts. MRM ion chro-
matographs for the pyrrole, lactam, and hydroxylactam adducts
in reaction mixtures containing Etn only, IsoK only, or both IsoK
and Etn. IsoK, ␥-ketoaldehyde isomer; MRM, multiple reaction
monitoring.
incubation at the various IsoK concentrations, suggesting
that the majority of IsoK reacted with cellular amines. Lev-
els of both protein and PE adducts in HEK293 cells in-
creased as the concentration of IsoK in the treatment
media increased (Fig. 9A). Under these conditions, the
average amount of measured PE and protein adducts rep-
resented 6.9% and 1.7%, respectively, of the reacted IsoK,
consistent with the greater reactivity rate of PE.
To determine whether IsoK-PE adducts were also
formed in cell types relevant to disease, we measured the
extent of IsoK-PE formation in human umbilical vein en-
Fig. 8. Streptomyces chromofuscus PLD hydrolyzes
IsoK-PE to IsoK-Etn. IsoK-PE preparation was treated
with vehicle or S. chromofuscus PLD for 2 h, IsoK-[2H4]
Etn added as internal standard, and the resulting
products analyzed by LC/MS/MS for IsoK-Etn. Left
panel: MRM ion chromatographs for reaction with
PLD only. Middle panel: MRM ion chromatographs
for IsoK-PE treated with vehicle only. Right panel:
MRM ion chromatographs for PLD treated IsoK-PE.
IsoK, ␥-ketoaldehyde isomer; MRM, multiple reac-
tion monitoring; PE, phosphatidylethanolamine;
PLD, phospholipase D.
Cytotoxic isoketal PE adducts
1007

Fig. 10. IsoK-PE is cytotoxic to HUVEC. PE was treated with ei-
ther 0.1 molar equivalent of IsoK (IsoK-PE), the inactive dimethoxy
acetal synthetic precursor of IsoK (DMA-PE), or the DMSO vehicle
(veh-PE) and 0.1 to 30 ␮M of the resulting PE reaction mixture
incubated with HUVEC overnight. Viability was determined by
MTT conversion assay and normalized to viability of untreated
cells. HUVEC, human umbilical vein endothelial cell; IsoK,
␥-ketoaldehyde isomer; PE, phosphatidylethanolamine.
is the active mediator of some of the biological effects of
IsoK. Our finding that IsoK-PE alone is sufficient to induce
cytotoxicity in HUVEC is consistent with the notion that
IsoK induces cytotoxicity at least in part by forming IsoK-PE
which then directly induces cell death. While in vitro stud-
ies clearly indicate that direct covalent modification of en-
zymes by IsoK can profoundly alter enzymatic activity, the
biological function of membrane associated enzymes and
receptors could also be altered by noncovalent interac-
tions with IsoK-PE. Modification of the terminal amine of
PE by the bulky and acidic IsoK would be anticipated to
significantly disrupt inner leaflet membrane packing and
curvature and thereby perturb membrane integrity and
membrane protein function. Such potential perturbations
of mitochondrial membranes by IsoK-PE might plausibly
underlie the cytotoxicity to HUVEC induced by IsoK-PE.
IsoK-PE might also act as ligand for receptors, analogous
to recent findings that N-acyl-PE suppresses food intake
via cFOS expression in neuropeptide Y neurons (24) and
suppresses phagocytosis via Rac1 and Cdc42 in mac-
rophages (25). The activation of a membrane receptors
linked to cell death by IsoK-PE would be consistent with
the previously described activation of p38-MAPK by IsoK
in platelets (16). While no receptors for lipid modified PE
have been identified to date, several G-protein coupled re-
ceptors and nuclear receptors bind lipids as high affinity
ligands and the physiological ligands for many receptors
remain uncharacterized, so that future studies may well
identify specific lipid modified PE receptors.
Fig. 9. PE adducts form to a greater extent than protein ad-
ducts in cells. IsoK was added for 2 h at increasing concentra-
tions to HEK293 or HUVEC cells plated in six well plates and
the amount of PE and protein adduct quantified by LC/MS/MS
and normalized to amount of DNA in each well. A: Levels of PE
and protein adduct in HEK293 cells incubated with 0-10 ␮M
IsoK. B: Levels of PE and protein adduct in HUVEC cells incu-
bated with 0-1 ␮M IsoK. IsoK, ␥-ketoaldehyde isomer; PE,
phosphatidylethanolamine.
DISCUSSION
While the formation of protein adducts by IsoK has
been well established in vivo and presumed to mediate the
biological effects of IsoK, the extent of IsoK adduction to
other biologically relevant amines in vivo and their bio-
logical activity remains poorly characterized. Our findings
suggest that at least one other type of adduct, PE adducts,
are formed in greater abundance in cells and may mediate
some of the effects of IsoK. While our findings were being
revised for publication, Li et al. published a report finding
IsoK modified PE in human plasma, as well as in the liver
of ethanol treated mice (27). Interestingly, the levels of PE
adducts that they reported in healthy volunteers (3.4 ng/
ml) are substantially higher than the levels of protein ad-
duct than we have previously reported in plasma (0.56 ng/
ml) (17). The plasma ratio of PE to protein adducts in
these two studies are very similar to the ratio of PE to pro-
tein adducts that we found in our cellular studies. Another
publication during the revision period by Carrier et al.
showing that IsoK formed adducts on deoxycytidine (28)
is consistent with our finding that IsoK also formed DNA
adduct to a lesser extent than protein adducts.
One general characteristic of receptor ligands is the ex-
istence of mechanisms to regulate their levels, particularly
degradative enzymes. The finding that the PLD from S.
chromofuscus can cleave IsoK-PE in addition to N-acyl-PE
suggests that lipases active against N-acyl-PE in other spe-
cies might also be able to degrade IsoK-PE. Of interest in
this regard is the recent finding that in mammalian cells
there are at least two enzymes, N-acyl-PE -PLD (29) and
The finding that PE adducts are formed to a greater ex-
tent than protein adducts raises the possibility that IsoK-PE
1008
Journal of Lipid Research Volume 51, 2010

E2-protein adducts in human plasma and vasculature. Chem. Res.
Toxicol. 10: 536–545.
12. Davies, S. S., M. Talati, X. Wang, R. L. Mernaugh, V. Amarnath,
J. Fessel, B. O. Meyrick, J. Sheller, and L. J. Roberts II. 2004.
Localization of isoketal adducts in vivo using a single-chain anti-
body. Free Radic. Biol. Med. 36: 1163–1174.
13. Talati, M., B. Meyrick, R. S. Peebles, Jr., S. S. Davies, R. Dworski,
R. Mernaugh, D. Mitchell, M. Boothby, L. J. Roberts II, and J. R.
Sheller. 2006. Oxidant stress modulates murine allergic airway re-
sponses. Free Radic. Biol. Med. 40: 1210–1219.
14. Poliakov, E., M. L. Brennan, J. Macpherson, R. Zhang, W. Sha, L.
Narine, R. G. Salomon, and S. L. Hazen. 2003. Isolevuglandins, a
novel class of isoprostenoid derivatives, function as integrated sen-
sors of oxidant stress and are generated by myeloperoxidase in
vivo. FASEB J. 17: 2209–2220.
15. Hoppe, G., G. Subbanagounder, J. O’Neil, R. G. Salomon, and
H. F. Hoff. 1997. Macrophage recognition of LDL modified by
levuglandin E2, an oxidation product of arachidonic acid. Biochim.
Biophys. Acta. 1344: 1–5.
16. Bernoud-Hubac, N., D. A. Alam, J. Lefils, S. S. Davies, V. Amarnath,
M. Guichardant, L. J. Roberts Ii, and M. Lagarde. 2009. Low concen-
trations of reactive [gamma]-ketoaldehydes prime thromboxane-
dependent human platelet aggregation via p38-MAPK activation.
Biochim. Biophys. Acta. 1791: 307–313.
17. Brame, C. J., O. Boutaud, S. S. Davies, T. Yang, J. A. Oates, D.
Roden, and L. J. Roberts II. 2004. Modification of proteins by
isoketal-containing oxidized phospholipids. J. Biol. Chem. 279:
13447–13451.
18. Davies, S. S., V. Amarnath, K. S. Montine, N. Bernoud-Hubac, O.
Boutaud, T. J. Montine, and L. J. Roberts II. 2002. Effects of re-
active gamma-ketoaldehydes formed by the isoprostane pathway
(isoketals) and cyclooxygenase pathway (levuglandins) on protea-
some function. FASEB J. 16: 715–717.
19. Davies, S. S. 2008. Modulation of protein function by isoketals and
levuglandins. Subcell. Biochem. 49: 49–70.
20. Amarnath, V., K. Amarnath, K. Amarnath, S. Davies, and L. J.
Roberts II. 2004. Pyridoxamine: an extremely potent scavenger of
1,4-dicarbonyls. Chem. Res. Toxicol. 17: 410–415.
21. Bernoud-Hubac, N., L. B. Fay, V. Armarnath, M. Guichardant, S.
Bacot, S. S. Davies, L. J. Roberts II, and M. Lagarde. 2004. Covalent
binding of isoketals to ethanolamine phospholipids. Free Radic.
Biol. Med. 37: 1604–1611.
22. Davies, S. S., E. J. Brantley, P. A. Voziyan, V. Amarnath, I. Zagol-
Ikapitte, O. Boutaud, B. G. Hudson, J. A. Oates, and L. J. Roberts
II. 2006. Pyridoxamine analogues scavenge lipid-derived gamma-
ketoaldehydes and protect against H(2)O(2)-mediated cytotoxic-
ity. Biochemistry. 45: 15756–15767.
23. Hidalgo, F. J., F. Nogales, and R. Zamora. 2004. Determination
of pyrrolized phospholipids in oxidized phospholipid vesicles and
lipoproteins. Anal. Biochem. 334: 155–163.
24. Gillum, M. P., D. Zhang, X-M. Zhang, D. M. Erion, R. A. Jamison,
C. Choi, J. Dong, M. Shanabrough, H. R. Duenas, D. W. Frederick,
et al. 2008. N-acylphosphatidylethanolamine, a gut- derived circu-
lating factor induced by fat ingestion, inhibits food intake. Cell.
135: 813–824.
␣/beta-hydrolase 4 (30), that hydrolyze N-acyl-PE. It would
be of great interest to determine if IsoK-PE is a substrate
for either of these enzymes and the effect of genetic
ablation of these enzymes on endothelial cell viability
or inflammatory signaling during oxidative stress and
inflammation.
Given the greater abundance of PE compared with pro-
tein adducts, it is of interest to consider whether the ef-
fects of IsoK scavengers such as salicylamine, which protects
cells from hydrogen peroxide induced cytotoxicity (22),
might be mediated at least in part by protecting against
the formation of IsoK-PE adducts. Future studies will be
need to determine both the mechanisms of this cytotoxic-
ity and if other cell types are also vulnerable.
In summary, our finding that IsoK-PE is one of the most
abundant adducts of IsoK in cells identifies a new poten-
tial mediator of the effects of IsoK and provides methods
to examine the extent of its formation in physiologically
relevant conditions.
The authors appreciate the valuable advice of Dr. Stephen
Milne on mass spectrometric analysis of phospholipids and
Pablo Darelli for excellent technical assistance.
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