Oxidation of plasmalogen, low-density lipoprotein and raw 264.7 cells by photoactivatable atomic oxygen precursors

Article abstract of DOI:10.1111/php.12201

The oxidation of lipids by endogenous or environmental reactive oxygen species (ROS) generates a myriad of different lipid oxidation products that have important roles in disease pathology. The lipid oxidation products obtained in these reactions are dependent upon the identity of the reacting ROS. The photoinduced deoxygenation of various aromatic heterocyclic oxides has been suggested to generate ground state atomic oxygen (O[³P]) as an oxidant; however, very little is known about reactions between lipids and O(³P). To identify lipid oxidation products arising from the reaction of lipids with O(³P), photoactivatable precursors of O( ³P) were irradiated in the presence of lysoplasmenylcholine, low-density lipoprotein and RAW 264.7 cells under aerobic and anaerobic conditions. Four different aldehyde products consistent with the oxidation of plasmalogens were observed. The four aldehydes were: tetradecanal, pentadecanal, 2-hexadecenal and hexadecanal. Depending upon the conditions, either pentadecanal or 2-hexadecenal was the major product. Increased amounts of the aldehyde products were observed in aerobic conditions. The photodeoxygenation of dibenzothiophene S-oxide has been suggested to generate ground state atomic oxygen (O[³P]). The reactivity of the putative O(³P) is distinct from other reactive oxygen species (ROS) and little is known about its reactivity with lipids and other biomolecules. In this work, exposure of low-density lipoprotein (LDL) to O(³P) yielded four aldehyde products. The same aldehydes were observed after the oxidation of an isolated plasmalogen by O(³P).

Full text of DOI:10.1111/php.12201

Photochemistry and Photobiology, 2014, 90: 386–393
Oxidation of Plasmalogen, Low-Density Lipoprotein and RAW 264.7 Cells
by Photoactivatable Atomic Oxygen Precursors†
Max T. Bourdillon1, Benjamin A. Ford2, Ashley T. Knulty3, Colleen N. Gray1, Miao Zhang1,
David A. Ford*2 and Ryan D. McCulla*1
1Department of Chemistry, Saint Louis University, St. Louis, MO
2Department of Biochemistry and Molecular Biology, and Center for Cardiovascular Research, Saint Louis University,
School of Medicine, St. Louis, MO
3Department of Chemistry, College of the Ozarks, Point Lookout, MO
Received 14 August 2013, accepted 24 October 2013, DOI: 10.1111/php.12201
ABSTRACT
The oxidative species generated during photodeoxygenation of
aromatic heterocyclic oxides undergoes reactions consistent with
what would be expected for O(3P). For example, the putative
O(3P) reacts with O2 to form O3, selectively oxidizes tertiary
hydrogen of alkanes, displays diffusion-limited rate constants of
a magnitude that would be expected for a very small oxidant and
yields similar product ratios compared to O(3P) produced by
microwave discharge (9,11–13). However, due to the difficulties
in directly detecting O(3P) in solution, definitive evidence for
freely diffusing O(3P) in these photochemical reactions has not
been achieved.
Regardless of the exact nature of the oxidant, the reactivity
profile of the putative O(3P) is distinct from other ROS. For
example, comparison of reaction rate constants for a series of
compounds for reactions between the putative O(3P) and hydro-
xyl radical found O(3P) was considerably more selective than
hydroxyl radical. In this previous study, reaction rate constants
ranged over three orders of magnitude for O(3P), whereas for
hydroxyl radical, the reaction rate constants were nearly identical
for the same series of compounds (11). More recently, the photo-
deoxygenation of 2,8-hydroxymethyl-dibenzothiophene S-oxide
(1) was shown to more easily oxidize thiols than other reactive
functional groups in aqueous solutions (13). This suggested
O(3P) could be used to selectively oxidize cysteine residues over
other amino acids within a protein. The photodeoxygenation of 1
resulted in the near quantitative oxidation of a critical cysteine
residue of Arabidopsis thaliana adenosine-5′-phosphosulfate
kinase (14). This observation was consistent with the expected
selectivity of O(3P) as the use of most other ROS would be
expected to result in more promiscuous oxidation of other amino
acid residues.
The oxidation of lipids by endogenous or environmental reac-
tive oxygen species (ROS) generates a myriad of different
lipid oxidation products that have important roles in disease
pathology. The lipid oxidation products obtained in these
reactions are dependent upon the identity of the reacting
ROS. The photoinduced deoxygenation of various aromatic
heterocyclic oxides has been suggested to generate ground
state atomic oxygen (O[3P]) as an oxidant; however, very little
is known about reactions between lipids and O(3P). To
identify lipid oxidation products arising from the reaction of
lipids with O(3P), photoactivatable precursors of O(3P) were
irradiated in the presence of lysoplasmenylcholine, low-
density lipoprotein and RAW 264.7 cells under aerobic and
anaerobic conditions. Four different aldehyde products con-
sistent with the oxidation of plasmalogens were observed. The
four aldehydes were: tetradecanal, pentadecanal, 2-hexadece-
nal and hexadecanal. Depending upon the conditions, either
pentadecanal or 2-hexadecenal was the major product.
Increased amounts of the aldehyde products were observed in
aerobic conditions.
INTRODUCTION
Oxidative damage caused by reactive oxygen species (ROS) has
long been implicated to have an important role in human disease
and aging (1). Despite their toxicity, increasing evidence indi-
cates nature has evolved to use ROS as important secondary
messengers in cell signaling (2). In addition, the reactive nature
of ROS has been exploited to probe the structure and function of
biomolecules in techniques such as chromophore-assisted ligand
inactivation and hydroxyl radical footprinting (3–5). The diverse
roles played by ROS highlights the importance of understanding
their fundamental reactivity with biomolecules.
While the irradiation of O(3P)-precursors has been shown to
selectively oxidize cysteine residues in proteins and cause single
strand-scission in DNA (14–16), very little is known about the
oxidation of lipids by O(3P). The oxidation of lipids by ROS
results in a variety of lipid oxidation products that have been
implicated as mediators of human disease. The alkene bonds of
polyunsaturated fatty acids and sterol ring of cholesterol are tar-
gets for ROS. These oxidized lipid species are produced during
inflammation in vascular walls and thus participate in atheroscle-
rosis, during infiltration of immune cells into ischemic and
ischemic reperfused tissues as well as into tumors. Furthermore,
Ground state atomic oxygen (O[3P]) is an important species in
atmospheric chemistry and has been postulated to form during
the photodeoxygenation of aromatic heterocyclic oxides (6–10).
*Corresponding authors email: rmccull2@slu.edu (R. McCulla) and fordda@slu.edu
(D. Ford)
†This paper is part of the Special Issue honoring the memory of Nicholas J. Turro.
© 2013 The American Society of Photobiology
386

Photochemistry and Photobiology, 2014, 90 387
Ozonolysis of pLPC and photolysis mixtures. Samples were exposed
to ozone by bubbling through a stream of ozone generated by an Ozotech
Poseidon ozone generator (Yreka, CA) being supplied with pure oxygen
for 10 min. For control experiments, a stream of oxygen was passed
through the samples for 10 min.
Quantification of aldehyde products from pLPC oxidation. Samples
were directly analyzed by capillary column gas chromatography–elec-
tron impact mass spectrometry (EI-GC-MS). The GC-MS analysis was
lipids in the lung are exposed to environmental oxidants and
ROS produced by subsequent inflammatory responses to environ-
mental-elicited lung injury.
Plasmalogens are a class of lipids that are particularly sensi-
tive to oxidation by ROS due their vinyl ether linkage to the
glycerol backbone. The vinyl ether bond has been shown to have
a higher reactivity with a variety of ROS including ozone, per-
oxyl radicals, superoxide and HOBr/HOCl (17–21). Gas phase
reaction between O(3P) and alkenes are known to occur rapidly,
and the reaction between styrene and O(3P) in acetonitrile yields
styrene oxide and phenylacetaldehyde as products (12). Given
the sensitivity of plasmalogens to oxidation, it was hypothesized
that plasmalogens would be sensitive to oxidation by O(3P) and
yield distinct oxidation products. By pursuing this hypothesis, it
was sought to determine if O(3P) could be used to generate
unique lipid oxidation products in a biological sample.
carried out using
a Shimadzu 30 m (0.25 mm ID 9 0.25 lm film
thickness) SHRXI-5MS column attached to Shimadzu GCMS-QP2010S
mass spectrometer. Helium was used as the carrier gas at a flow rate of
1.0 mL minÀ1, and the column temperature was initially set at 100°C
for 3 min, then increased by 10°C minÀ1 until 250°C, and then held at
250°C for 10 min. The ion source temperature was set to 200°C and
the electron energy to 70 eV. Standard curves were made using the
authentic samples of the detected aldehydes using dodecane as an inter-
nal standard.
Irradiation of LDL and 2,8-hydroxymethyl-dibenzothiophene S-oxide
(1). A 100 lL aliquot from the solution containing 0.63 mg mLÀ1 of
LDL was added to 1.5 mL of water containing 1 mM of 1. Four drops of
1-octanol was added to samples to decrease foaming while samples were
degassed. Additions of more than four drops of 1-octanol resulted in the
formation of precipitate during sparging, and thus, careful addition of the
1-octanol was required. The samples were then degassed by sparging
with argon for 30 min. The samples were then loaded onto a rotating
merry-go-round apparatus, and irradiated in a Luzchem LZC-4C photore-
actor using eight broadly emitting fluorescent bulbs centered at 350 nm
for 4 h.
RAW 264.7 cell studies. RAW 264.7 cells were seeded on six-well
34.6-mm-diameter cell culture plates in high glucose DMEM supple-
mented with 10% FBS. Prior to experimental conditions, cell culture
media was removed and then 1 mL of a 2 mM solution of 1 in PBS or
PBS without 1 was added to the cells. The plates were then incubated for
25 min. The plates were then irradiated with a Luzchem LZC-4C with
14 Hitachi FL8BL-B broadly emitting fluorescent bulbs centered at
350 nm bulbs for 2 h.
Lipid Extraction for LDL and RAW-264.7 cells. The media was spun
down to remove any cell fragments and collected for media analysis.
Cells were scraped twice with 0.75 mL 154 mM saline solution. The
cells and media, as well as LDL samples, were extracted using the
Bligh and Dyer lipid extraction technique in the presence of 5 pmol
of 2-[d5]-hexadecenal (27). Extraction was conducted three times per
sample. After lipid extraction, samples underwent silica extraction on
LC-Si (Supelco) columns using chloroform solvent to separate plasmal-
ogens from free fatty aldehydes (28). The samples were then prepared
for GC-MS analysis by derivatization with pentafluorobenzyl (PFB)
hydroxylamine. Derivatization with PFB hydroxylamine was performed
by resuspending the reaction products in 300 lL of ethanol and
300 lL of PFB hydroxylamine (6 mg mLÀ1) in water. The ethanol–
water mixture was vortexed for 5 min at room temperature with fur-
ther incubation at room temperature for 25 min. Following the 25 min
incubation, 1.2 mL of water was added to the reaction products, and
the reaction products were extracted with 4:1 v:v cyclohexane/diethyl
ether followed by resuspension in petroleum ether prior to GC-MS
analysis.
Aldehyde quantification for LDL and RAW-264.7 cells. After derivati-
zation with PFB hydroxylamine, the samples were analyzed by capillary
gas chromatography–mass spectrometry (GC-MS). GC-MS analysis was
performed in the negative ion chemical ionization mode (NICI) with
methane as the reagent gas using a DB-1 column (12 m, 0.2 mm inner
diameter, 0.33 mm methyl silicone film coating: P.J. Cobert), and a 6890
gas chromatograph-5973 mass spectrometer (Agilent). The source temper-
ature was set at 150°C. The electron energy was 193.3 eV and the emis-
sion current was 49.4 A. The injector and transfer lines were maintained
at 250°C and 280°C, respectively. The GC oven was maintained at
150°C for 3.5 min, increased at the rate of 25°C minÀ1 to 310°C and
held at 310°C for another 5 min. Quantification of 2-hexadecenal was
performed utilizing selected ion monitoring (SIM) by comparing the inte-
grated area corresponding to m/z = 413 to that produced from the deuter-
ated internal standard 2-[d5]-hexadecenal at m/z = 418. Similarly,
hexadecanal and pentadecanal were monitored by SIM at m/z = 415 and
m/z = 401, respectively. Standard curves were made using the authentic
samples of the detected aldehydes using 2-[d5]-hexadecenal as an internal
standard.
MATERIALS AND METHODS
Materials. Dibenzothiophene S-oxide (DBTO) was prepared by the oxi-
dation of dibenzothiophene (DBT) with mCPBA by a slight modification
to a previously reported procedure (see Supporting Information) (22).
2,8-hydroxymethyl-dibenzothiophene S-oxide (1) was prepared from
DBT by previously described procedures (13). Authentic tetradecanal
(TDA), pentadecanal (PDA) and hexadecanal (HDA), were all purchased
from TCI America and used as received. Authentic 2-hexadecenal
(2-HDEA) was purchased from Toronto Research Chemicals and 2-
[15,15,16,16,16-d5]-hexadecenal (2-[d5]-hexadecenal) was prepared as
previously described (23).
Preparation of Lysoplasmenylcholine. The lysoplasmenylcholine
molecular species, 1-O-hexadec-1′-enyl-sn-glycero-3-phosphocholine
(pLPC), was prepared from bovine heart lecithin and purified as previ-
ously described (24). Following base methanolysis of bovine heart leci-
thin, the reaction product, lysoplasmenylcholine, was purified by HPLC,
and the lysoplasmenylcholine molecular species, pLPC, was subse-
quently purified by reversed-phase HPLC. Prepared pLPC was >95%
pure as determined by thin-layer chromatography, straight phase HPLC,
reversed-phase HPLC and capillary gas chromatography. pLPC was
subjected to acid methanolysis in the presence of arachidic acid (20:0
fatty acid) as an internal standard and quantified by capillary gas chro-
matography by comparisons of the integrated peak area of the dimethyl
acetal of palmitaldehyde (derived from the sn-1 aliphatic chain of
pLPC) to that of the methyl ester of arachidic acid (derived from the
internal standard).
LDL isolation from peripheral blood. Human blood was collected for
the preparation of LDL as authorized by Saint Louis University Institu-
tional Review Board Protocol 10014. LDL from peripheral blood was
isolated as described previously (25). In brief, whole blood (50 mL)
was taken from healthy human volunteers and anticoagulated with
EDTA (final concentration, 5.4 mM) before centrifugation at 500 9 gmax
for 20 min at 24°C. The resulting supernatant was collected and
adjusted to a density of 1.019 g mLÀ1 by the addition of KBr and then
ultracentrifuged at 388 000 9 gmax for 8 h at 4°C without braking.
After removing the top VLDL layer, the density of the remaining sam-
ple was adjusted to 1.063 g mLÀ1 by the addition of KBr, and then it
was ultracentrifuged again under the conditions specified previously.
The yellow lipemic layer floating in the top half of the tube was col-
lected and desalted using PD10 columns. LDL protein concentration
was assayed by a Markwell-modified Lowry assay (26).
Irradiation of DBTO and pLPC. A 50 lL aliquot from a solution con-
taining pLPC dissolved in chloroform (9.41 mg mLÀ1) was added to a
fused-silica quartz test tube. The chloroform was then removed with a
gentle stream of nitrogen. The pLPC was then reconstituted with 1.5 mL
of acetonitrile (HPLC grade) containing 1.0 mM of DBTO and 1.0 mM of
dodecane as an internal standard. The test tubes were then sealed with a
rubber septa, and for anaerobic samples, the samples were sparged with
argon for 30 min. The samples were then loaded onto a rotating merry-
go-round apparatus, and irradiated in a Luzchem LZC-4C photoreactor
using eight broadly emitting fluorescent bulbs centered at 350 nm for
5 h.

388 Max T. Bourdillon et al.
RESULTS AND DISCUSSION
The photodeoxygenation of dibenzothiophene S-oxide (DBTO)
has been suggested as a clean method of generating O(3P)
(8,29). As side reactions are common with other possible precur-
sors of O(3P), DBTO was selected as the photoactivatable source
of O(3P). However, the aqueous solubility of DBTO is low, and
thus, other aqueous soluble precursors of O(3P), such as 2,8-hy-
droxymethyl-dibenzothiophene S-oxide (1), have been recently
developed (13). As shown in Scheme 1, DBTO was used as the
photoactivatable O(3P)-precursor in acetonitrile, and 1 was used
for generating O(3P) in aqueous solutions.
The toxicity of lipid oxidation products has been thoroughly
demonstrated and reviewed over the past few decades (30–32).
For example, 4-hydroxynonenal has been shown to demonstrate
cytotoxic and carcinogenic effects at doses of <1 lM (33,34).
Lipid oxidation products are often the result of the oxidation of
unsaturated fatty acids. Likewise, unsaturated fatty acids were
the expected targets for oxidation by O(3P) as O(3P) reacts with
alkenes with rate constants on the order of 1 9 109 M sÀ1 in
acetonitrile (11,12). To examine the reaction of O(3P) with a
variety of different lipids quickly, low-density lipoprotein was
irradiated in the presence of 2,8-hydroxymethyl-dibenzothioph-
ene S-oxide (1).
Scheme 1. Photodeoxygenation of DBTO and 2,8-hydroxymethyl-
dibenzothiophene Soxide (1).
Irradiation of LDL and 2,8-hydroxymethyl-dibenzothiophene
S-oxide (1) yields aldehydes consistent with the oxidation of
plasmalogens
Low-density lipoprotein (LDL) from human blood was chosen as
a model system. The predominant protein in LDL is apolipopro-
tein B. Also, LDL is comprised of phospholipids on the surface
of the lipoprotein protocol and a central lipid core containing tri-
glycerides and cholesteryl esters. Furthermore, approximately
40% of the esterified fatty acid in LDL is polyunsaturated (35).
Aqueous solutions containing 0.063 mg mLÀ1 and 1 mM of 1
were irradiated with UV-A light (350 nm). After irradiation, the
samples were derivatized with PFB hydroxylamine and then
analyzed by GC-MS. As shown in Scheme 2, the PFB deriva-
tives of four aldehydes were observed: tetradecanal (TDA), pen-
tadecanal (PDA), 2-hexadecenal (2-HDEA) and hexadecanal
(HDA), which were identified by comparison to the PFB deriva-
tives of authentic aldehydes.
The detected aldehyde products were suspected of arising
from the oxidation of plasmalogens within the LDL particles.
The amount of plasmalogens present in the fatty acid pools is
variable in different tissues and has been estimated to represent
as much as 12% of all phospholipids (36). Plasmalogens are par-
ticularly sensitive to oxidation due to their vinyl ether linkage to
the glycerol backbone, which has been shown to have a higher
reactivity with a variety of ROS (17–21). The products obtained
from the oxidation of plasmalogens are dependent upon the
ROS. For example, long-chain aldehydes were observed after
plasmalogens in Chinese hamster ovary cells were exposed to
singlet oxygen. The oxidative cleavage of the vinyl ether p-bond
was proposed to proceed through the decomposition of a dioxe-
tane intermediate (37). Conversely, exposure of low and high-
density lipoprotein to hydroxyl radical resulted in the formation
of a-hydroxy aldehydes (38,39). Also, Wynalda and Murphy
have shown that the ozonolysis of plasmalogen yields long-
chain, saturated aldehydes and plasmalogens were considerably
Scheme 2. Oxidation of LDL by irradiated 1.
more sensitive to ozonolysis than other unsaturated fatty acids
(19).
Like other ROS, direct oxidation of the vinyl ether linkage by
O(3P) was expected to yield long-chain aldehydes. As mentioned
above, plasmalogens are sensitive to ozonolysis, and thus, in aer-
obic conditions, the formation of O3 from the reaction of O(3P)
and O2 could potentially account for some observed aldehydes
such as TDA, PDA and HDA. Oxidation of other monounsatu-
rated or polyunsaturated fatty acids by O(3P) or O3 would be
expected to result in different aldehydes. For example, common
monounsaturated fatty acids such as palmitoleic acid (16:1 n-7)
or oleic (18:1 n-9) would be expected to yield heptanal or non-
anal, respectively, upon oxidative cleavage. Also, the ozonolysis
of common monounsaturated and polyunsaturated fatty acids are
not known to yield 2-HDEA.
The reaction between O2 and O(3P) yields O3. To examine
the role of O3 in aerobic conditions, LDL and 1 were irradiated
with UV-A light with and without prior sparging with argon.
Control experiments of samples without 1 (350 nm, in Fig. 1) or
without exposure to light (1, in Fig. 1) were also performed, and
it should be noted that measurable amounts of the observed
products were observed in these controls arising from sample
handling during PFB derivatization. The results of these experi-
ments are shown in Fig. 1. As shown in Fig. 1A, 2-HDEA and
PDA were the dominant products in aerobic conditions where
106 and 78 pmol per 0.1 mg of LDL was observed, respectively.
However, a significant change, compared to controls, was also
observed for TDA and HDA as well. If O3 was being generated

Photochemistry and Photobiology, 2014, 90 389
in appreciable amounts, it was expected that 2-HDEA would
react to form TDA. Thus, it was postulated that 2-HDEA was
the result of a direct reaction between O(3P) and the lipids, pos-
sibly a plasmalogen or another unsaturated fatty acid, within the
LDL.
1.3, 4.0 and 5.5 pmol per 0.1 mg of LDL for TDA, 2-HDEA
and HDA, respectively, could be claimed. This was somewhat
unexpected as 2-HDEA was expected to increase in the absence
of O3. In attempt to provide greater clarity for these unexpected
results, the oxidation of an isolated plasmalogen was pursued.
To examine how the removal of O2 from the reaction would
influence the observed products, samples were sparged with
argon prior to irradiation. To achieve anaerobic conditions,
extreme care was required as foaming was observed when a
gentle stream of argon gas was passed through the samples.
Foaming was greatly reduced by the addition of 1-octanol; how-
ever, except at very low concentrations of 1-octanol, the combi-
nation of sparging and 1-octanol resulted in the formation of a
precipitate that could not be dissolved even with sonication.
These difficulties resulted in a greater amount of error associated
with the data in anaerobic conditions. As shown in Fig. 1B,
decreased levels of all the aldehyde products were observed in
anaerobic conditions compared with aerobic conditions. Also, the
increase observed between the sample (1, 350 nm) and the con-
trols was considerably smaller. Compared to the irradiated con-
trol (350 nm), an increase of only 2.8, 10.0, 10.3 and 6.8 pmol
per 0.1 mg of LDL for TDA, PDA, 2-HDEA and HDA, respec-
tively, was observed. In addition, the increase in PDA was not
significant from the photocontrol when the standard error for the
measurements was considered. When taking account of the
uncertainty represented by the standard error, an increase of only
Aldehyde products from the oxidation of pLPC by O(3P)
To examine the reaction of O(3P) with plasmalogens, pLPC was
selected as a model plasmalogen. As shown in Scheme 3, irradi-
ation of a solution of DBTO in acetonitrile with UV-A light in
the presence of pLPC resulted in the formation of tetradecanal
(TDA), pentadecanal (PDA), hexadecanal (HDA) and 2-hexadec-
enal (2-HDEA). These were the only four aldehydes that were
detectable by GC-MS analysis. As described below, the identity
of the aldehyde products was determined by the comparison to
authentic samples of TDA, PDA, HDA and 2-HDEA. Compari-
son of the GC-MS results for authentic 2-HDEA and the putative
2-HDEA from the photolysis mixture is shown in Fig. 2. The
peak retention times for authentic 2-HDEA and the unknown
aldehyde were 14.88 and 14.89 min, respectively. Inspection of
the authentic 2-HDEA and unknown mass spectra revealed simi-
lar fragmentation patterns. Thus, the unknown aldehyde was ten-
tatively assigned to 2-HDEA. Identification of the remaining
TDA, PDA and HDA was performed in a similar fashion (see
Figs. S1–S3 in the Supporting Information).
Figure 1. Amount of aldehyde products observed upon irradiation of LDL and 1 with broadly emitting 350 nm bulbs (UV-A) in anaerobic and aerobic
conditions. (A) aerobic conditions, (B) anaerobic conditions. Error bars represent the standard error from three or more experiments. A Q-test with 99%
confidence was performed to remove outlying data due to gross errors.
Scheme 3. Oxidation of pLPC by irradiated DBTO.

390 Max T. Bourdillon et al.
(entry 2), a much smaller increase (~30 lM) of PDA was
observed, and the other aldehydes were either not detected or
showed no change. For both aerobic and anaerobic conditions,
samples that were not irradiated showed no formation of the
aldehyde products (entry 3 and 6). It should be noted that in all
samples, trace amounts of HDA were always detected during
GC-MS analysis. The other aldehydes products were not detect-
able prior to irradiation. Therefore, for HDA, the change in
HDA concentrations prior and after the experiments (entries 1–8)
is reported in Table 1.
Additional evidence for 2-hexadecenal
While GC-MS comparison to authentic 2-HDEA supported the
assignment of the unknown product as 2-HDEA, more evidence
was desired to rule out other possible potential isomers. To con-
firm the position of the carbon–carbon double bond, solutions of
pLPC (700 lM) and DBTO (1 mM) were irradiated with UV-A
light for 4 h, which as expected resulted in the formation of the
four different aldehyde products. The irradiated samples were
then exposed to ozone for 10 min. After ozonolysis, the alde-
hyde tentatively assigned to 2-HDEA was completely consumed,
and a 200 lM increase in both TDA and PDA was observed,
which were the expected ozonolysis products of 2-HDEA and
unreacted pLPC, respectively. No other new aldehyde products
were observed. To confirm TDA as the expected ozonolysis
product of 2-HDEA, a sample of 100 lM of authentic 2-HDEA
in acetonitrile was exposed to ozone in a similar fashion. Follow-
ing ozonolysis, no 2-HDEA was detectable by GC-MS, and the
only aldehyde observed was 80 Æ 30 lM of TDA. The absence
of shorter alkyl aldehydes after ozonolysis and nearly identical
GC-MS compared to authentic 2-HDEA provides strong evi-
dence that the assignment of the olefin containing aldehyde prod-
uct as 2-HDEA was correct.
In the aerobic conditions, significantly more of PDA and
2-HDEA, 630 Æ 230 lM and 80 Æ 30 lM, respectively, and
about double the amount of TDA and HDA, 13 Æ 4 lM and
4.5 Æ 1.7 lM, respectively, were produced upon irradiation of
the sample (entry 4). Unlike in anaerobic conditions, small
amounts of TDA and 2-HDEA were also detected in the irradi-
ated control (entry 5). The increase observed in aerobic condi-
tions was statistically significant in the total amount of aldehyde
being produced in aerobic conditions. The similar amount of
DBT being produced in anaerobic and aerobic conditions (entries
1 and 4) indicates the increase in amount of aldehyde being pro-
duced in aerobic conditions is not due to simply more oxidant
being produced. It is tempting to suggest that in aerobic condi-
tions that O3 results in greater oxidation of the vinyl ether link-
age, which has been shown to be very sensitive to ozonolysis
(19). However, the relative ratio of the two major aldehyde prod-
ucts PDA and 2-HDEA remained the same at 7.8:1 of PDA to
2-HDEA, respectively. As O3 reacts with pLPC to largely form
PDA (entry 7), the similar ratio of PDA to 2-HDEA in anaerobic
and aerobic conditions was counter to appreciable amounts of O3
being formed. If some of the O(3P) was being converted to O3,
an increase in the amount of TDA and PDA at the expense of 2-
HDEA would have been expected. This result does not rule out
the formation of O3 in these samples, but suggested that O3 was
not a significant fraction of the active oxygen species.
Quantitation of aldehyde products in aerobic and anaerobic
conditions
The reaction between molecular oxygen and O(3P) to form ozone
has a measured rate constant of 4 9 109 M sÀ1 in water (40).
Thus, in aerobic conditions, formation of O3 during the photo-
deoxygenation of DBTO and its analogs is possible. To investi-
gate the effect of removing oxygen, samples of 710 lM of pLPC
and 1 mM DBTO in acetonitrile were irradiated with and without
sparging with argon prior to irradiation. Formation of the alde-
hyde products and deoxygenation product dibenzothiophene
(DBT) are given in Table 1. When the samples were deoxygen-
ated, irradiation of the solution resulted in the formation of
220 Æ 90 lM of PDA and 28 Æ 8 lM of 2-HDEA and small
increases in TDA and HDA (entry 1). In the absence of DBTO
In the oxidation of LDL by 1, the observed aldehydes were
suggested to arise from the oxidation of plasmalogens by O(3P).
The significant formation of TDA, PDA and 2-HDEA during the
oxidation of pLPC by DBTO suggests this was plausible. The
Figure 2. Comparison GC-MS results of unknown product from pLPC oxidation by UV irradiation of DBTO to authentic sample of 2-hexadecenal.
(A) GC-MS of unknown aldehyde. (B) GC-MS data for an authentic sample of 2-hexadecenal.

Photochemistry and Photobiology, 2014, 90 391
Table 1. Amounts of pLPC aldehyde products produced during UV irradiation of DBTO and control experiments*.
Entry
Cond.
O2†
[DBT]‡
Total [CHO]‡
[TDA]‡
[PDA]‡
[2-HDEA]‡
[HDA]‡
1
2
3
4
5
6
7
8
DBTO§, hm¶
hm
DBTO
DBTO, hm
hm
DBTO
Ozone**
Air††
N
N
N
Y
Y
Y
–
470 Æ 170
250 Æ 100
26 Æ 11
6.7 Æ 2.3
n.d.‡‡
n.d.
13 Æ 4
2 Æ 3
n.d.
220 Æ 90
29 Æ 9
n.d
630 Æ 230
52 Æ 17
n.d.
28 Æ 8
n.d.
n.d
80 Æ 30
13 Æ 7
n.d.
2.8 Æ 1.7
À2 Æ 7
–
6.9 Æ 7.7
1.1 Æ 2.0
730 Æ 240
71 Æ 18
1.1 Æ 2.0
4.5 Æ 1.7
3.7 Æ 2.4
0.0 Æ 2.0
14 Æ 4
360 Æ 100
–
À2 Æ 4
0.0 Æ 2.0
730 Æ 260
0.6 Æ 1.2
–
–
15 Æ 5
700 Æ 190
n.d.
n.d.
–
n.d.
n.d.
0.6 Æ 1.2
*Samples of 653 lM of pLPC in 6 mL acetonitrile contained within fused-silica test tubes were used for all experiments. †(N) Oxygen removed from
samples by sparging with argon for 30 min. (Y) samples irradiated without sparging. ‡Concentrations in lM determined by GC-MS reported as
mean Æ 95% confidence interval for five or more experiments. A Q-test with 99% confidence was performed to remove outlying data due to gross
errors. §Samples Contains 1.0 mM of DBTO. ¶sample irradiated with eight broadly emitting fluorescent bulbs centered at 350 nm for 5 h. **Ozone gas
gently passed through sample for 10 min. ††Air gently passed through sample for 10 min. ‡‡not detected.
Scheme 4. Possible mechanism in aerobic conditions.
decrease in the amount of observed aldehyde products in deoxy-
genated samples was also consistent with the results obtained for
the oxidation of LDL. Therefore, the presence of O2 uniformly
increased the amount of aldehyde products being observed. How-
ever, as discussed above, the results were inconsistent with the
involvement of O3, and no evidence for the formation of singlet
oxygen during the irradiation of 1 in aerobic conditions has been
previously reported (13). An alternative explanation of the increase
in oxidation products in aerobic conditions is that O2 reacts with
intermediates formed after the reaction between O(3P) and the
plasmalogen. The epoxidation of styrene by O(3P) was suggested
previously to arise from diradical (12). Consistent with this
previously suggested mechanism, formation of ozonide B after the
reaction between the diradical A and O2 would be expected to
yield PDA as shown in Scheme 4 (p-addition pathway). Formation
of 2-HDEA could potentially arise from an allylic H-abstraction
leading to the allylic hydroperoxide D in the presence of oxygen,
which has been proposed to convert to 2-HDEA (37). While these
proposed mechanisms are consistent with previous suggested
mechanisms, the true mechanism and the role of O2 in the oxida-
tion of these lipids during UV-A irradiation of 1 and DBTO were
not resolved by the experiments reported here.
Exposure of RAW 264.7 cells to O(3P) does not result in the
formation aldehyde products
The predominant ether linked phospholipid in RAW 264.7 cells
is plasmenylethanolamine, which comprises about 36% of the
total ethanolamine glycerolipid pool (41). These cells were irradi-
ated with 350 nm light in 1 enriched media to test whether or
not plasmalogen oxidation would be observed in a more complex
sample. After extraction and PFB derivatization similar to the
LDL procedures, only trace amounts of the previously observed
aldehydes could be detected, and no statistically relevant change
compared to control samples that either were not irradiated or
contained no 1 was observed. Simply not generating enough oxi-
dant was a potential explanation of this observation. However,
the 2 mM starting concentration of 1 was expected to generate
approximately 800 lM of O(3P) under these conditions (14).
From previous work, 4-vinylbenzoic acid was not easily oxidized
by O(3P) in water, where as 3-mercaptobezonic acid was quanti-
tatively converted to the corresponding disulfide. Glutathione
(GSH) concentrations in cells can reach millimolar concentrations.
Thus, an alternative explanation was another antioxidant, such as
GSH, was more competitive for O(3P) than the plasmalogens

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Acknowledgements—Portions of this material are based on work
supported by the National Science Foundation under CHE-1255270. The
authors thank the National Science Foundation for support under grant
CHE-0963363 for renovations to the research laboratories in Monsanto
Hall. This work was additionally supported by donors to the Herman
Frasch Foundation and the Presidential Research Fund at Saint Louis
University.
SUPPORTING INFORMATION
Additional Supporting Information may be found in the online
version of this article:
A description of the preparation of DBTO and Figures S1–S3
can be found at DOI: 10.1562/2006-xxxxxx.s1.
Figure S1. Comparison GC-MS results of unknown product
from pLPC oxidation by UV irradiation of DBTO to authentic
sample of tetradecanal. A: GC-MS of unknown aldehyde. B:
GC-MS data for an authentic sample of tetradecanal.
Figure S2. Comparison GC-MS results of unknown product
from pLPC oxidation by UV irradiation of DBTO to authentic
sample of pentadecanal. A: GC-MS of unknown aldehyde. B:
GC-MS data for an authentic sample of pentadecanal.
Figure S3. Comparison GC-MS results of unknown product
from pLPC oxidation by UV irradiation of DBTO to authentic
sample of hexadecanal. A: GC-MS of unknown aldehyde. B:
GC-MS data for an authentic sample of hexadecanal.
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