Nonenzymatic free radical-catalyzed generation of 15-deoxy-Δ 12,14- Prostaglandin J2-like compounds (deoxy-J 2-isoprostanes) in vivo1

Article abstract of DOI:10.1194/jlr.M010264

15-Deoxy-δ^12,14 -prostaglandin J₂(15-d-PGJ ₂) is a reactive cyclopentenone eicosanoid generated from the dehydration of cyclooxygenase-derived prostaglandin D₂(PGD ₂). This compound possesses an α , β -unsaturated carbonyl moiety that can readily adduct thiol-containing biomolecules such as glutathione and cysteine residues of proteins via the Michael addition. Due to its reactivity, 15-d-PGJ₂is thought to modulate infl ammatory and apoptotic processes and is believed to be an endogenous ligand for peroxisome proliferator-activated receptor- γ . However, the extent to which 15-d-PGJ₂is formed in vivo and the mechanisms that regulate its formation are unknown. Previously, we have reported the formation of PGD ₂and PGJ₂-like compounds, termed D₂/J ₂-isoprostanes (D₂/J₂-IsoPs), produced in vivo by the free radical-catalyzed peroxidation of arachidonic acid (AA). Based on these fi ndings, we investigated whether 15-d-PGJ₂-like compounds are also formed via this nonenzymatic pathway. Here we report the generation of novel 15-d-PGJ₂-like compounds, termed deoxy-J₂- isoprostanes (deoxy-J₂-IsoPs), in vivo, via the nonenzymatic peroxidation of AA. Levels of deoxy-J₂-IsoPs increased 12-fold (6.4 ± 1.1 ng/g liver) in rats after oxidant insult by CCl 4 treatment, compared with basal levels (0.55 ± 0.21 ng/g liver). These compounds may have important bioactivities in vivo under conditions associated with oxidant stress. -Hardy, K. D., B. E. Cox, G. L. Milne, H. Yin, and L. Jackson Roberts II. Nonenzymatic free radical-catalyzed generation of 15-deoxy- δ^12,14- Prostaglandin J2-like compounds (deoxy-J2-isoprostanes) in vivo. by the American Society for Biochemistry and Molecular Biology, Inc.

Full text of DOI:10.1194/jlr.M010264

Nonenzymatic free radical-catalyzed generation of
1
2,14
1
(
5-deoxy-⌬ -prostaglandin J -like compounds
2
1
deoxy-J -isoprostanes) in vivo
2
,
†
Klarissa D. Hardy,* Brian E. Cox,* Ginger L. Milne,* Huiyong Yin,* and
2
, ,†
L. Jackson Roberts II *
†
Division of Clinical Pharmacology,* and Department of Pharmacology, Vanderbilt University School of
Medicine, Nashville, TN 37232
1
2,14
Abstract 15-Deoxy-⌬
-prostaglandin J (15-d-PGJ ) is a
Cyclooxygenase 1 (COX-1) and COX-2 catalyze the
committed step in the formation of prostaglandins (PG)
from arachidonic acid (AA) by generating the unstable
2
2
reactive cyclopentenone eicosanoid generated from the
dehydration of cyclooxygenase-derived prostaglandin D2
PGD ). This compound possesses an ␣,␤-unsaturated car-
bonyl moiety that can readily adduct thiol-containing biomol-
ecules such as glutathione and cysteine residues of proteins
via the Michael addition. Due to its reactivity, 15-d-PGJ is
(
2
bicyclic endoperoxide intermediate, PGH (1–3). Tissue-
2
specific synthases convert PGH to the parent PGs PGF ,
2
2␣
PGE , PGD , PGI , and thromboxane (TXA ) (1). These
2
2
2
2
2
molecules are formed in response to various stimuli, and
they exert a plethora of biological effects through interac-
tion with cell surface receptors (1).
thought to modulate inflammatory and apoptotic processes
and is believed to be an endogenous ligand for peroxisome
proliferator-activated receptor-␥. However, the extent to
which 15-d-PGJ is formed in vivo and the mechanisms that
PGE and PGD are unstable and readily undergo dehy-
2
2
2
regulate its formation are unknown. Previously, we have re-
ported the formation of PGD and PGJ -like compounds,
dration in an aqueous solution to form cyclopentenone
PGs PGA and PGJ , respectively. Dehydration of PGE re-
2
2
2
2
2
termed D /J -isoprostanes (D /J -IsoPs), produced in vivo
2
2
2
2
sults in formation of PGA (4). Dehydration of PGD re-
2
2
by the free radical-catalyzed peroxidation of arachidonic
acid (AA). Based on these findings, we investigated whether
sults in the formation of PGJ (5). PGJ can isomerize in
2
2
1
2
vitro in the presence of albumin to ⌬ -PGJ and then un-
2
1
5-d-PGJ -like compounds are also formed via this non-
2
12,14
dergo dehydration to yield 15-deoxy-⌬ -PGJ (15-d-PGJ )
enzymatic pathway. Here we report the generation of novel
5-d-PGJ -like compounds, termed deoxy-J -isoprostanes
2
2
1
(
(Fig. 1) (5). Unlike other classes of PGs, cyclopentenone
PGs are characterized by the presence of an electrophilic
␣,␤-unsaturated carbonyl moiety in the prostane ring, ren-
dering them susceptible to Michael addition with nucleo-
philicbiomolecules, suchasthefreesulfhydrylsofglutathione
2
2
deoxy-J -IsoPs), in vivo, via the nonenzymatic peroxidation
2
of AA. Levels of deoxy-J -IsoPs increased 12-fold (6.4 ± 1.1
2
ng/g liver) in rats after oxidant insult by CCl treatment,
4
compared with basal levels (0.55 ± 0.21 ng/g liver). These
compounds may have important bioactivities in vivo under
conditions associated with oxidant stress.—Hardy, K. D., B.
E. Cox, G. L. Milne, H. Yin, and L. Jackson Roberts II. Nonen-
zymatic free radical-catalyzed generation of 15-deoxy-⌬12,14-
prostaglandin J2-like compounds (deoxy-J2-isoprostanes) in
vivo. J. Lipid Res. 2011. 52: 113–124.
(
GSH) and cysteine residues of proteins (6). These com-
pounds have attracted considerable attention because they
exhibit a unique spectrum of biological activities, presum-
ably through reaction with cellular biomolecules (6).
1
5-d-PGJ , one of the most well-defined cyclopentenone
2
Supplementary key words 15-d-PGJ • cyclopentenone eicosanoid •
2
lipid peroxidation • oxidant stress • isoprostane
Abbreviations: AA, arachidonic acid; AAPH, 2,2′-azobis
amidinopropane)dihydrochloride; APCI, atmospheric pressure chemi-
(
cal ionization; CID, collision-induced dissociation; COX, cyclooxygenase;
GSH, glutathione; GST, glutathione S-transferase; HPLC, high-perfor-
mance liquid chromatography; IsoPs, isoprostanes; NF-␬B, nuclear
factor kappa B; PC, phosphatidylcholine; PG, prostaglandin; PLA2, phos-
This research was supported by National Institutes of Health Grants GM15431
and ES13125. Its contents are solely the responsibility of the authors and do not
necessarily represent the official views of the National Institutes of Health.
K.D.H. is supported by National Institutes of Health Ruth L. Kirschstein Na-
tional Research Service Award 1F31AG035483-01 from the National Institute
of Aging.
pholipase A ; PPAR-␥, peroxisome proliferator-activated receptor-␥; RT,
2
retention time; SIM, selected ion monitoring; SRM, selected reaction
monitoring; TXA , thromboxane.
2
1
This article is dedicated to the memory of Dr. Jason Morrow and to
his mentorship and scientific guidance in the studies described
herein.
Manuscript received 2 August 2010 and in revised form 7 October 2010.
2
Published, JLR Papers in Press, October 13, 2010
DOI 10.1194/jlr.M010264
To whom correspondence should be addressed.
email: jack.roberts@vanderbilt.edu
Copyright © 2011 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 52, 2011
113

Fig. 1. 15-d-PGJ2 is formed from PGD2 through a
sequence of dehydration and isomerization reactions.
PGs, is particularly reactive because it possesses two elec-
trophilic carbon centers that are susceptible to nucleo-
philic attack: one center is in the cyclopentenone ring
series regioisomers depending on the carbon atom to
which the side chain hydroxyl is attached (20).
The extent to which cyclopentenone prostanoids are
generated in vivo has been the subject of continuing con-
troversy for the last 3 decades (21–24). We have previously
(
C-9), and the other is on the lower side chain (C-13). This
compound is believed to mediate a number of cellular re-
sponses through covalent interaction with critical intracel-
reported that, analogous to the formation of PGA and
2
lular protein targets (7, 8). 15-d-PGJ is postulated to be an
PGJ from the dehydration of COX-generated PGE and
2
2
2
endogenous ligand of peroxisome proliferator-activated
receptor-␥ (PPAR-␥), which plays an important role in adi-
pocyte differentiation and antiinflammatory processes (9).
PGD , respectively, cyclopentenone IsoPs (A /J -IsoPs) are
2
2
2
also formed in vivo from the dehydration of E /D -IsoPs
2
2
(25). However, evidence for the formation of 15-d-PGJ in
2
1
5-d-PGJ has also been shown to exhibit antiinflamma-
vivo is lacking. Hirata et al. (21) reported that the precur-
2
1
2
tory, antiproliferative, and proapoptotic activities inde-
sor of 15-d-PGJ , ⌬ -PGJ , can be detected in human urine
2 2
pendent of PPAR-␥ in studies performed in vitro. However,
and that its formation is suppressed to some extent by
the extent to which 15-d-PGJ is formed in vivo and the
COX inhibitors. Levels of free 15-d-PGJ excreted in hu-
2
2
mechanisms regulating its formation remain unclear.
Oxidant stress has been increasingly implicated in the
pathogenesis of a number of human diseases, including
atherosclerosis, cancer, neurodegenerative disorders, and
even the normal aging process (10–13). Lipid peroxida-
tion is a central feature of oxidant stress. We have previ-
ously reported discovery of PG-like compounds, termed
isoprostanes (IsoP), which are produced in vivo from free
radical-induced peroxidation of AA, independent of COX
man urine were found to be very low (22). Heretofore, it
has been assumed that synthesis of 15-d-PGJ depends
2
upon COX generation of PGD . Given their structural and
2
functional similarity, it is highly likely that similar to the
dehydration of COX-derived PGD , D -IsoPs can undergo
2
2
dehydration and isomerization to yield a series of 15-d-
PGJ -like compounds, termed deoxy-J -IsoPs. A proposed
2
2
mechanism for formation of the four regioisomers of
deoxy-J -IsoPs is shown in Fig. 2. It should be noted that dehy-
2
(
14). IsoPs are isomeric to PGs, differing in the stereo-
dration of the 15- series D -IsoPs followed by isomerization
2
chemical relationships of the two side chains on the pros-
tane ring. PG side chains are in the trans configuration
with respect to the prostane ring, whereas the majority of
IsoP side chains are in the cis configuration. Additionally,
unlike PGs, IsoPs are initially formed in situ in phospho-
lipids and are then subsequently hydrolyzed to their free
form by phospholipases (15, 16). The mechanism for the
formation of IsoPs from AA has been well defined. Follow-
ing abstraction of a bisallylic hydrogen from AA and the
addition of a molecule of oxygen to form a peroxyl radical,
the radical undergoes 5-exo cyclization, and a second mol-
ecule of oxygen is added to the backbone of the compound
of the 13,14 double bond would result in loss of the chiral
center at C-12, eliminating the stereochemical orientation
of the lower side chain that distinguishes IsoPs from PGs.
A single chiral center would be retained at C-8. Thus, a
compound identical in all respects to 15-d-PGJ and its cor-
2
responding enantiomer would be expected to form from
dehydration and isomerization of 15-D -IsoP (Fig. 3). For
2
purposes of discussion hereafter, the PG possessing a
structure identical to that generated by COX is referred to
as 15-d-PGJ . The compound that is enantiomeric to COX-
2
derived 15-d-PGJ is referred to as ent-15-d-PGJ . The race-
2
2
mic mixture is termed rac-15-d-PGJ . Dehydration of other
2
to form PGG -like compounds (17, 18). These unstable
regioisomers of D -IsoPs yields various regioisomers of
2
2
bicycloendoperoxide intermediates can be reduced to
PGF -like compounds, termed F -IsoPs (14), or they un-
deoxy-J -IsoPs (Fig. 2).
Herein, we report that 15-d-PGJ2 and 15-d-PGJ -like
2
2
␣
2
2
dergo rearrangement to PGE - and PGD -like compounds
compounds (deoxy-J -IsoPs) are formed in significant
2
2
2
(
E /D -IsoPs) (19) or TXA -like compounds (Tx-IsoPs).
abundance in vitro and in vivo via the nonenzymatic free
radical-catalyzed peroxidation of AA, independent of COX
2
2
2
Based on this mechanism of formation, four regioisomers
of each class of IsoPs derived from AA are generated, each
of which can theoretically be composed of eight racemic
diastereomers. Compounds are denoted 5-, 8- 12-, or 15-
enzymes. The finding that a series of deoxy-J -IsoPs are
2
generated in vivo through lipid peroxidation is of poten-
tial biological importance due to the predicted bioactivity
1
14
Journal of Lipid Research Volume 52, 2011

Fig. 2. Proposed pathway for the formation of the deoxy-J -IsoPs via the nonenzymatic peroxidation of AA. For simplicity, the stereo-
2
chemistry of both the lipid peroxidation intermediates and final IsoPs are not shown.
of these compounds. Similar to 15-d-PGJ , each of the
chromatography (HPLC) columns were purchased from Phenom-
enex (Torrance, CA). All solvents were of HPLC quality and were
purchased from EM Science (Gibbstown, NJ).
2
deoxy-J -IsoPs possesses reactive electrophilic carbon
2
centers, which render them susceptible to nucleophilic
addition reactions with thiol-containing biomolecules,
such as GSH and cysteine residues of proteins. Because
Oxidation of AA in vitro
Five mg of AA was dissolved in 50 ␮l of ethanol and added im-
mediately to 4.95 ml of phosphate-buffered saline (PBS) (pH 7.4)
containing 10 mM 2,2′-azobis(amidinopropane)dihydrochloride
the biological effects of 15-d-PGJ are often likely to be me-
2
diated by the compound’s reactivity with cellular thiols,
all of the deoxy-J -IsoPs would be expected to exert simi-
2
lar biological effects in vivo under settings of oxidant
stress.
MATERIALS AND METHODS
Chemical reagents and supplies
Human serum albumin, CCl , triphenyl-phosphine (TPP), and
4
Apis mellifera venom phospholipase A2 (PLA ) were purchased
2
from Sigma-Aldrich. PGD , 15-d-PGJ , and isotopically labeled 15-
2
2
2
d-PGJ ([ H ]15-d-PGJ ) was purchased from Cayman Chemical
2
4
2
Co. (Ann Arbor, MI). Arachidonic acid was purchased from NU-
Check Prep, Inc. (Elysian, MN). The C18 Sep-Paks were purchased
from Waters Corporation (Milford, MA). High-performance liquid
Fig. 3. Structures of rac-15-d-PGJ , consisting of 15-d-PGJ and its
enantiomer.
2
2
Formation of deoxy-J -IsoPs in vivo
115
2

(
AAPH), a free radical initiator. The AA oxidation reaction mix-
under 1.5 mTorr of argon. Spectra shown were obtained at 24 eV.
Spectra were displayed by averaging scans across chromato-
graphic peaks. Selected reaction monitoring (SRM) was per-
formed according to the characteristic fragmentation patterns of
ture was incubated at 37°C for 24 h. Following oxidation, excess
triphenylphosphine (TPP) was added to the oxidized AA mix-
ture (1 mg/ml) for 5 min at room temperature to reduce hy-
droperoxides to hydroxyls. Subsequently, the oxidized AA sample
was extracted twice with 2 ml of ethyl acetate, and extracts were
dried under nitrogen gas. Samples were stored in 200 ␮l of etha-
nol at Ϫ80°C until analyzed by LC/tandem mass spectrometry
15-d-PGJ determined by CID. The collision energy for SRM was
2
24 eV. Data acquisition and analysis were performed using Xcali-
ber version 2.0 software.
(MS/MS).
Adduction of deoxy-J -IsoPs with glutathione in vitro
2
To isolate deoxy-J -IsoPs in vitro, AA was oxidized as described
above and extracted with ethyl acetate. The sample was dried under
2
Isolation of 15-d-PGJ -like compounds (deoxy-J -IsoPs)
2
2
from rat liver
a stream of N and reconstituted in 100 ␮l of ethanol. This sample
2
CCl (1 mg/kg body weight in 1 ml of corn oil) was adminis-
was dissolved in 0.5 ml of 1 N HCl and incubated for 5 h at 37°C
to facilitate acid-catalyzed dehydration of D /J -IsoPs to deoxy-J -
IsoPs. Following incubation, the reaction mixture was diluted to
10 ml with 50 mM aqueous ammonium acetate (pH 3.4) and ex-
tracted three times with 1.5–2 ml of ethyl acetate. Extracts were
4
tered orogastrically to male Sprague-Dawley rats to induce lipid
peroxidation (14). Control animals received corn oil alone. Four
hours after treatment, the animals were anesthetized with pento-
barbital (60 mg/kg, intraperitoneally) and euthanized. Livers
were removed and immediately flash-frozen in liquid nitrogen
and stored at Ϫ80°C. Tissues samples weighing 300–500 mg were
homogenized in 5 ml of ice-cold chloroform-methanol (2:1, v/v)
containing butylated hydroxytoluene (0.005%) to prevent ex
2
2
2
pooled, dried under a stream of N , and reconstituted in 100 ␮l of
2
ethanol. This sample was dissolved in 1 ml of PBS (pH 6.5) and
incubated in the presence of excess reduced GSH (ف1 mg) and
ف1 mg of equine liver glutathione S-transferase (GST) for 2 h at
37°C. A similar method was used for the incubation of chemically
vivo autooxidation. Esterified deoxy-J -IsoPs in liver phospholip-
2
ids were enzymatically hydrolyzed with A. mellifera venom PLA to
synthesized 15-d-PGJ in the presence of GSH and GST. After the
2
2
liberate free deoxy-J -IsoPs. Subsequently, free IsoPs were ex-
incubation, GSH adducts of deoxy-J -IsoPs and synthesized 15-d-
2
2
tracted over a C18 Sep-Pak cartridge that had been precondi-
tioned by rinsing with methanol and deionized water, pH 3. The
C18 Sep-Pak cartridge was rinsed with 10 ml each of deionized
water (pH 3) and heptane, and the sample was eluted with 10 ml
of 1:1 ethyl acetate-heptane. The solvent was removed by evapo-
PGJ were purified by extraction using a C Sep-Pak cartridge pre-
conditioned with 10 ml of acetonitrile and 50 mM aqueous
ammonium acetate (pH 3.4) and eluted with 10 ml of 95% etha-
2
18
nol, and dried under a stream of N . Samples were suspended in a
2
3:1 mixture of Phase 1-Phase 2 (Phase 1, water-phase 2-acetic acid,
95:5:0.1; Phase 2, acetonitrile-methanol-acetic acid, 95:5:0.1), and
ration under a stream of N , and samples were stored in 200 ␮l of
2
ethanol at Ϫ80°C until LC/MS/MS analysis.
deoxy-J -IsoP-GSH adducts were definitively identified by LC/
2
ESI-MS/MS. The reverse-phase HPLC conditions described above
were used. For MS/MS experiments, CID analysis of the parent
ion m/z 622 of 15-d-PGJ -GSH adduct and putative deoxy-J -IsoP-
GSH adducts was performed at 18 to 22 eV under 1.5 mTorr of
argon. Spectra shown were obtained at 22 eV.
Analysis of 15-d-PGJ -like compounds (deoxy-J -IsoPs)
2
2
by LC/MS/MS
2
2
After oxidized AA was extracted by using the C18 Sep-Pak
method described above, the sample was suspended in 100 ␮l of
2
:1 methanol-water, and deoxy-J -IsoPs were analyzed by reverse-
2
phase HPLC. Online HPLC was carried out using a Surveyor MS
pump equipped with a Phenomenex Luna C18 column (50 × 2.00
mm, 3 ␮m) at a flow rate of 0.3 ml/min starting with 80% Phase
RESULTS
1
(water-phase 2-acetic acid, 95:5:0.1) to 30% from 1 to 27.0 min
Formation and characterization of 15-d-PGJ and
2
and holding at 100% Phase 2 (acetonitrile-methanol-acetic acid,
1
5-d-PGJ -like compounds in vitro
2
9
3
5:5:0.1) from 28.0 to 29.0 min and returning to 80% Phase 1 at
0.0 to 32.0 min. LC/electrospray ionization (ESI)-MS/MS was
A representative selected ion monitoring (SIM) chro-
matogram obtained from LC/MS/MS analysis of chemi-
carried out with a ThermoFinnigan TSQ/Quantum Ultra mass
spectrometer. The ESI source was fitted with a deactivated fused
silica capillary (100-␮m inner diameter), and the mass spectrom-
eter was operated in the negative ion mode. Nitrogen was used as
both the sheath gas and the auxiliary gas, at 31 and 17 psi, respec-
tively. The capillary temperature was 300°C. The spray voltage
was 5.0 kV, and the tube lens voltage was 80 V. In another set of
experiments, deoxy-J -IsoPs were analyzed by normal phase
HPLC/atmospheric pressure chemical ionization (APCI)-MS for
separation of deoxy-J -IsoP regioisomers. For these experiments,
deoxy-J -IsoPs were extracted by C18 Sep-Pak chromatography as
cally synthesized 15-d-PGJ is shown in Fig. 4A (upper
2
chromatogram). When analyzed by ESI-MS in the negative
ion mode, the predicted precursor ion for 15-d-PGJ is m/z
2
3
15. The peak eluting at 19.4 min was analyzed by CID,
and predicted fragments were obtained (Fig. 4A, lower
Ϫ
spectrum). These included m/z 271 [M Ϫ CO ] and m/z
2
2
2
03 (Fig. 4A, lower spectrum). The smaller peak in the
upper chromatogram is likely a synthetic isomer of 15-d-
2
PGJ as results from LC/ESI-MS/MS analysis of this peak
2
2
described previously, and samples were resuspended in mobile
phase (93:7, hexane-isopropanol-acetic acid) for normal phase
HPLC. Online HPLC was carried out using a Waters Alliance
HPLC equipped with a Phenomenex Luna silica column (250 ×
were identical to that of the authentic 15-d-PGJ2.
AA was then oxidized in vitro using the free radical ini-
tiator AAPH, and extracts were analyzed for 15-d-PGJ and
2
other 15-d-PGJ -like compounds (deoxy-J -IsoPs). When ana-
2
2
4.60 mm, 5 ␮m) at a flow rate of 1.0 ml/min with an isocratic
lyzed by LC/ESI-MS/MS in negative ion mode, the predicted
gradient of 93:7:0.1, hexane-isopropanol-acetic acid for 20 min.
LC/APCI-MS was carried out with a ThermoFinnigan TSQ/Quan-
tum Ultra mass spectrometer operating in the negative ion mode
with an APCI source. MS/MS analyses using collision-induced dis-
sociation (CID) of the precursor ion m/z of 315 of putative
precursorionfordeoxy-J -IsoPsism/z315.TheSIMchromato-
2
gram of this ion is shown in Fig. 4B. As is evident, multiple
chromatographic peaks are present that presumably repre-
sent different deoxy-J -IsoP regioisomers obtained from the
2
1
5-d-PGJ and deoxy-J -IsoPs were performed from 18 to 27 eV
nonenzymatic peroxidation of AA. The chromatographic
2
2
1
16
Journal of Lipid Research Volume 52, 2011

Fig. 4. LC/ESI-MS/MS analysis of 15-d-PGJ (A) and a mixture of 15-d-PGJ -like compounds (deoxy-J -
2
2
2
IsoPs) obtained from the nonenzymatic oxidation of AA in vitro (B). A: LC/ESI-MS/MS analysis of a chemi-
cally synthesized 15-d-PGJ employing SIM (upper chromatogram) and CID (lower product ion spectrum).
2
B: LC/ESI-MS/MS analysis of 15-d-PGJ -like compounds obtained from the in vitro oxidation of AA employ-
2
ing SIM for the precursor ion m/z 315 (upper chromatogram) and CID (lower product ion spectrum) indic-
ative of putative 15-d-PGJ2.
peak eluting at 20.0 min has nearly the same retention
structural information, and the CID spectrum is shown in
time as authentic 15-d-PGJ . An authentic sample of isoto-
Fig. 4B. The observed product ions, m/z 271 and m/z 203,
2
2
pically labeled 15-d-PGJ2 ([ H ]15-d-PGJ ) showed the
are consistent with fragmentation of 15-d-PGJ . The series
4
2
2
identical retention time upon co-injection (see below).
Analyses described in legends to Fig. 4A and B were per-
formed on separate days, accounting for differences in LC
retention time.) This peak was analyzed by CID to obtain
of compounds representing deoxy-J -IsoPs elute in multi-
ple chromatographic peaks at approximately 19–22 min.
CID analysis of this series of chromatographic peaks supports
2
(
the contention that these are 15-d-PGJ -like compounds
2
Formation of deoxy-J -IsoPs in vivo
117
2

derived from different IsoP regioisomers, including com-
pounds of the 5-, 8-, 12-, and 15-series (Fig. 5). The com-
posite CID spectrum obtained by summing scans over the
PGJ generated from the nonenzymatic oxidation of AA,
2
and these compounds coelute with d -15-d-PGJ . Addi-
4
2
tional support for the identification of these compounds
entire series of chromatographic peaks reveals the prod-
as 15-d-PGJ and ent-15-d-PGJ was obtained through LC/
ultraviolet (UV) analysis, which showed that these com-
2
2
Ϫ
uct ion m/z 271 [M Ϫ CO ] as well as product ions result-
2
ing from specific fragmentation of each of the four
pounds possess a UV absorbance maximum (␭max) of 306
deoxy-J -IsoPs regioisomers, including m/z 217 (5-series),
nm, identical to that of authentic 15-d-PGJ (data not
2
2
m/z 95 (8-series), m/z 175 (12-series), and m/z 203 (15-
shown). To assess the total amount of deoxy-J -IsoPs
2
series). Additionally, we used normal phase HPLC to obtain
formed from the nonenzymatic oxidation of AA, LC/MS
quantification was performed by measuring the abun-
chromatographic separation of each of the deoxy-J -IsoPs
2
regioisomers. Shown in Fig. 6 is a representative chro-
dance of compounds representing deoxy-J -IsoPs relative
2
matogram of the different regioisomers of deoxy-J -IsoPs
to that of the d -15-d-PGJ internal standard. The mass
2
4
2
analyzed by normal phase LC/APCI-MS/MS. Again, CID
analysis of the series of chromatographic peaks is consis-
spectrometer was operated in SRM mode in the negative
ion mode, monitoring the precursor-to-product transition
tent with fragmentation of each of the deoxy-J -IsoP regio-
from m/z 315 to m/z 271 for the deoxy-J -IsoPs and moni-
2
2
isomers (Fig. 6).
toring the parent-to-product transition from m/z 319 to
m/z 275 for d -15-d-PGJ . Deoxy-J -IsoPs are generated in
significant abundance from the nonenzymatic oxidation
of AA in vitro, and levels are 16.0 ± 3.4 ng/mg AA (n ꢀ 4).
In additional studies, when we oxidized a phospholipid
containing sn-2-AA (palmitoyl-arachidonyl-phosphatidyl-
Due to their nonenzymatic formation, compounds gen-
erated from the free radical-catalyzed peroxidation of AA
would be predicted to be racemic. Thus, 15-series deoxy-J2-
IsoPs are predicted to comprise a racemic mixture of a
compound identical in all respects to COX-derived 15-d-
PGJ and its enantiomer, ent-15-d-PGJ . The racemic mix-
4
2
2
choline [PAPC]) in vitro, we found that 15-d-PGJ was
2
2
2
ture is referred to as rac-15-d-PGJ (Fig. 3). To confirm the
formed on the intact phospholipid (data not shown).
2
formation of rac-15-d-PGJ from the nonenzymatic oxida-
tion of AA, we used LC/MS/MS analysis with SRM. The
2
Adduction of deoxy-J -IsoPs with GSH in vitro
2
major product ion unique to rac-15-d-PGJ is m/z 203; thus,
One of our primary interests in the formation of 15-d-
PGJ and cyclopentenone deoxy-J -IsoPs in vivo is related
to the fact that these molecules contain electrophilic
␣,␤-unsaturated carbonyl moieties which render them sus-
ceptible to Michael addition with cellular nucleophiles.
2
we monitored the precursor-to-product transition of m/z
2
2
2
3
15 to m/z 203 to selectively detect rac-15-d-PGJ . [ H ]15-
2
4
d-PGJ (d -15-d-PGJ ) was used as an internal standard for
2
4
2
these analyses. The precursor ion of d -15-d-PGJ is m/z
4
2
319 due to the presence of four deuterium atoms on the up-
Because the biological effects of 15-d-PGJ are thought to
2
per side chain of the molecule, and CID analysis of d -15-
be mediated largely through its interaction with cellular
4
Ϫ
d-PGJ yields the major product ions m/z 275 [M Ϫ CO ]
thiols, all of the deoxy-J -IsoPs would be expected to exert
2
2
2
and m/z 203 (data not shown). Shown in Fig. 7 is a repre-
sentative chromatogram for the SRM analysis of rac-15-d-
similar biological effects through adduction to cellular
thiols. Thus, as a further characterization of these com-
Fig. 5. LC/ESI-MS/MS analysis of multiple regioisomers of deoxy-J -IsoPs generated from the nonenzymatic oxidation of AA in vitro. A:
2
Structurally specific fragmentation of the different deoxy-J -IsoP regioisomers. B: SIM chromatogram of the precursor ion at m/z 315 (up-
2
per) and CID spectrum (lower) obtained by summing scans over the chromatographic peaks, revealing structurally specific fragmentation
of the different deoxy-J -IsoP regioisomers.
2
1
18
Journal of Lipid Research Volume 52, 2011

Fig. 6. Normal phase LC/APCI-MS/MS analysis of multiple regioisomers of deoxy-J -IsoPs generated from
2
the nonenzymatic peroxidation of AA in vitro. SIM chromatogram of the parent ion at m/z 315 (upper) and
CID spectrum (lower) obtained by summing scans over the series of chromatographic peaks reveals structur-
ally specific fragmentation of the different deoxy-J -IsoP regioisomers.
2
pounds, we sought to determine whether deoxy-J -IsoPs
subjected to LC/MS as described above. Putative deoxy-J2-
IsoP-GSH adducts were definitively identified by LC/
ESI-MS/MS. The predicted precursor ion for deoxy-J2-
IsoP-GSH adducts in negative ion mode is m/z 622. SIM
analysis showed multiple peaks, with this m/z eluting from
10.7 to 15.3 min, presumably representing the GSH ad-
2
would adduct GSH, an abundant cellular thiol, in the
presence of GST. We have previously shown that 15-d-PGJ2
and other cyclopentenone eicosanoids readily adduct
GSH in the presence of GST in vitro (26–30). In the pres-
ent study, we incubated deoxy-J -IsoPs generated from the
2
nonenzymatic oxidation of AA in vitro with GSH in the
presence of GST. Adducts were partially purified from
an incubation mixture by C18 Sep-Pak extraction and
ducts of different deoxy-J -IsoP regioisomers (Fig. 8A).
These adducts elute at a retention time similar to that of the
2
GSH adduct of 15-d-PGJ . CID experiments confirmed
2
Fig. 7. Analysis of putative rac-15-d-PGJ in vitro by LC/MS/MS using SRM. LC/MS/MS analysis of rac-15-
2
2
d-PGJ (A) from the nonenzymatic oxidation of AA in vitro and chemically synthesized [ H ]15-d-PGJ (d -
2
4
2
4
1
5-d-PGJ ) (B). The mass spectrometer was operated in negative ion mode using SRM to monitor the
2
precursor-to-product transition of m/z 315–203 for rac-15-d-PGJ (chromatogram A) and the precursor-to-
2
product transition of m/z 319–203 for d -15-d-PGJ (chromatogram B).
4
2
Formation of deoxy-J -IsoPs in vivo
119
2

Fig. 8. Analysis of GSH adducts of deoxy-J -IsoPs and 15-d-PGJ by LC/ESI-MS/MS. A: SIM chromatogram
2
2
of putative deoxy-J -IsoP-GSH adducts with the precursor ion m/z 622. B: Composite CID spectrum obtained
2
by summing scans over the series of chromatographic peaks representing deoxy-J -IsoP-GSH adducts. C: CID
2
spectrum of the synthesized 15-d-PGJ -GSH adduct.
2
the molecules at m/z 622 to be deoxy-J -IsoP-GSH adducts.
IsoPs are initially formed in situ, esterified in phospholip-
ids, and hydrolyzed to their free form by platelet-activating
factor acetylhydrolase and possibly other phospholipases
2
A composite CID spectrum was obtained by summing
Ϫ
scans over these peaks (Fig. 8B). The precursor ion, M , is
present at m/z 622. Relevant product ions include m/z 306
(15). To quantify F -IsoPs esterified in tissues, the tissue is
2
Ϫ
[
M Ϫ deoxy-J -IsoP] (loss of the prostanoid portion of
initially treated with base to release F -IsoPs, which are
2
2
Ϫ
the molecule); m/z 272 [M Ϫ 350] (retro-Michael frag-
mentation); and m/z 254 [272 Ϫ H O] . A similar CID
then quantified. However, base treatment will induce de-
hydration of D -IsoPs to form J -IsoPs. Therefore, to quan-
Ϫ
2
2
2
spectrum was observed when chemically synthesized 15-d-
tify esterified deoxy-J -IsoPs formed in tissue, the tissue was
2
PGJ was incubated with GST and GSH (Fig. 8C). It should
be noted that the major product ions generated from
subjected to enzymatic hydrolysis using bee venom PLA2.
We found that this method of hydrolysis results in <0.3%
dehydration of PGD to 15-d-PGJ . We have previously
2
CID analysis of deoxy-J -IsoP-GSH adducts were indistin-
2
2
2
guishable from those generated from the CID analysis of
shown that treatment of rats with CCl induces a dramatic
4
the synthetic 15-d-PGJ -GSH adduct. Together, these data
increase in esterified IsoPs in the liver (19, 25). Therefore,
2
confirm that the molecules putatively identified as cyclo-
we examined whether deoxy-J -IsoPs are formed in vivo es-
2
pentenone deoxy-J -IsoPs are reactive and capable of un-
terified in liver phospholipids in rats that had been treated
2
dergoing nucleophilic addition reactions with GSH.
with CCl . Shown in Fig. 9 is a representative chromato-
4
gram from SRM analysis of deoxy-J -IsoPs (Fig. 9A), includ-
ing rac-15-d-PGJ2 (Fig. 9B), generated in vivo in liver
2
Isolation and quantification of deoxy-J -IsoPs
2
from rat liver
phospholipids in the rat after CCl treatment. Again, the
4
After confirming the formation of rac-15-d-PGJ and de-
entire series of deoxy-J -IsoPs was quantified by LC/ESI-
2
2
oxy-J -IsoPs via the nonenzymatic oxidation of AA in vitro,
we explored whether these compounds are formed in vivo.
MS using SRM as described previously. Levels of deoxy-J2-
IsoPs in vivo esterified in liver phospholipids in control
2
1
20
Journal of Lipid Research Volume 52, 2011

Fig. 9. Analysis of putative deoxy-J -IsoPs in vivo by LC/MS/MS using SRM. LC/MS/MS analysis of (A)
2
deoxy-J -IsoPs regioisomers and (B) the 15-series deoxy-J -IsoPs (rac-15-d-PGJ ) generated in vivo esterified
2
2
2
2
in liver phospholipids in rats after treatment with CCl and (C) chemically synthesized [ H ]15-d-PGJ (d -
4
4
2
4
1
5-d-PGJ ). The mass spectrometer was operated in the negative ion mode with SRM for the precursor-to-
2
product transitions of m/z 315–271 for all deoxy-J -IsoPs regioisomers (chromatogram A), m/z 315–203 for
2
rac-15-d-PGJ (chromatogram B), and m/z 319–275 for d -15-d-PGJ (chromatogram C).
2
4
2
rats were near the limit of detection (0.55 ± 0.21 ng/g
tissue; n ꢀ 5) for this method. However, in animals treated
formation have been unclear. In the present study, we have
shown for the first time the formation of 15-d-PGJ and 15-d-
2
with CCl , levels increased by ف12-fold to 6.4 ± 1.1 ng/g
PGJ -like compounds in vivo via free radical-catalyzed lipid
4
2
tissue (n ꢀ 5; P < 0.0001 compared with control). Addition-
ally, we have identified phosphatidylcholine (PC) contain-
ing esterified 15-d-PGJ (15-d-PGJ -PC) generated in vivo
peroxidation, independent of the COX pathway. Our results
suggest that a second pathway exists for the biosynthesis of
2
2
15-d-PGJ in vivo, and these findings provide further insight
2
in rats after oxidant insult (data not shown). These find-
into the biochemical mechanisms that can contribute to the
endogenous generation of reactive lipid mediators.
15-d-PGJ2 was originally identified as a dehydration
ings provide evidence that deoxy-J -IsoPs are produced in
2
significant abundance in vivo as products of free radical-
induced lipid peroxidation.
product of COX-derived PGD (5). Our initial interest in
2
determining whether 15-d-PGJ and 15-d-PGJ -like com-
2
2
pounds are formed via the free radical-catalyzed pathway
emerged from the observation that cyclopentenone PGA2
and PGJ -like compounds (A /J -IsoPs) are generated in
DISCUSSION
2
2
2
We report finding that a series of 15-d-PGJ -like com-
vivo in significant quantities esterified in liver phospholip-
ids in rats following oxidant insult (25). In addition, re-
cent studies by our laboratory support the contention that
PGs can be generated through the IsoP pathway, indepen-
dent of COX enzymes (31, 32). Gao et al. (31) reported
the formation of PGE and PGD and their respective
2
pounds, termed deoxy-J -IsoPs, are formed in vitro and in
2
vivo via free radical-catalyzed peroxidation of AA. Among
the deoxy-J products formed during this process, the
2
1
5-series deoxy-J -IsoPs (rac-15-d-PGJ ) comprises a race-
2
2
mic mixture of a molecule identical in all respects to 15-d-
2
2
PGJ and its corresponding enantiomer. The formation of
enantiomers in vitro and in vivo through the nonenzy-
matic peroxidation of AA by epimerization of E /D -IsoPs,
2
1
5-d-PGJ and 15-d-PGJ -like compounds in vivo is biologi-
2 2
2
2
cally relevant because 15-d-PGJ is thought to be an im-
specifically 15-E -IsoP and 15-D -IsoP, respectively. More
2
2t 2c
portant lipid mediator. Despite significant interest in the
recently, Yin et al. (32) reported the formation of PGF2␣ in
vivo via the IsoP pathway and demonstrated that the majority
of putative PGF2␣ in human urine is derived from free
bioactivity of 15-d-PGJ , the extent to which this compound
2
is formed in vivo and the mechanisms that regulate its
Formation of deoxy-J -IsoPs in vivo
121
2

radical-initiated peroxidation of AA, independent of COX
to occur through covalent binding of 15-d-PGJ to a criti-
2
enzymes. In the present study, we have found that J -IsoPs
cal cysteine residue in the PPAR-␥ ligand binding pocket
2
derived from D -IsoPs can also undergo further dehydra-
(42). Additionally, 15-d-PGJ has been shown to inhibit the
2
2
tion and isomerization in vivo to yield the cyclopentenone
deoxy-J -IsoPs. Dehydration of the 15-series J -IsoPs yields
a compound identical in all respects to COX-derived 15-d-
inflammatory response in a number of cell types, includ-
ing monocytes and macrophages, by blocking nuclear
factor kappa B (NF-␬B)-dependent gene expression via co-
valent modification of a critical cysteine residue in I␬B
kinase and the DNA-binding domains of NF-␬B subunits
2
2
PGJ and its enantiomer.
2
Cyclopentenone eicosanoids as a class have received
considerable attention over the past 3 decades due their
reactivity (6, 33). These compounds are characterized by
the presence of an electrophilic ␣,␤-unsaturated carbonyl
moiety in the prostane ring. This functional group renders
them susceptible to nucleophilic addition reactions with
thiol-containing biomolecules, which has been attributed
(38–41). 15-d-PGJ is also thought to be involved in the
2
resolution of inflammation by enhancing apoptosis in ac-
tivated macrophages through a p38 mitogen-activated pro-
tein kinase (MAPK)-dependent increase in reactive oxygen
species formation (38). Furthermore, 15-d-PGJ activates
2
the antioxidant response pathway via covalent modifica-
tion of reactive cysteine residues in Keap1, leading to Nrf2
activation and induction of antioxidant proteins, includ-
ing HO-1, peroxiredoxin 1, ␥-GCL, and heat shock pro-
to their biological effects. 15-d-PGJ , the most extensively
2
studied compound of the cyclopentenone eicosanoids, is
particularly reactive due to the presence of two electro-
philic carbons. The presence of two electrophilic centers
tein 70 (43–47). Finally, 15-d-PGJ has been reported to
2
in 15-d-PGJ is thought to contribute to its reactivity and
inhibit proliferation of vascular smooth muscle cells by in-
ducing cell cycle arrest (48, 49).
2
biological potency which are increased compared with
those of other cyclopentenone eicosanoids (34). The find-
ing that in addition to 15-d-PGJ , a series of deoxy-J -IsoPs
The finding that 15-d-PGJ and other deoxy-J -IsoPs are
2
2
generated in vivo via free radical-induced lipid peroxida-
tion has important implications for the pathophysiological
2
2
are formed in significant abundance in vivo is notable due
to the fact that these compounds also possess the reactive
electrophilic carbon centers that render them susceptible
to reaction with cellular nucleophiles. Like other IsoPs gen-
erated via the nonenzymatic peroxidation of AA, deoxy-J2-
IsoPs are formed as a series of four regioisomers, each
role of these compounds. Generation of 15-d-PGJ via the
2
COX pathway has been associated with settings of inflam-
mation (50–52). In the present study, we provide evidence
that 15-d-PGJ and 15-d-PGJ -like compounds, the deoxy-
2
2
J2-IsoPs, are generated in vivo under conditions of oxidant
stress. As a result of being generated through the COX
and free radical-catalyzed pathways, these compounds may
have additive effects in the pathophysiology of diseases,
such as atherosclerosis, in which inflammation and oxi-
dant stress are considered to play a critical role (53–56).
consisting of a mixture of stereoisomers. Deoxy-J -IsoPs
2
are derived from the dehydration and isomerization of the
different D -IsoP and J -IsoP regioisomers, including the
2
2
5
-, 8-, 12-, and 15-series. Of note, the 15-series deoxy-J2-
IsoPs comprise a racemic mixture of 15-d-PGJ and its en-
2
antiomer and are termed rac-15-d-PGJ . Consistent with
Previous studies have shown that 15-d-PGJ is synthesized
2
2
the presence of the polyunsaturated carbonyl moiety in
during mammalian inflammatory responses, and its pro-
duction may represent a feedback mechanism for the reso-
each of the deoxy-J -IsoPs, we have shown that these com-
2
pounds are reactive and readily undergo nucleophilic ad-
dition with GSH. Because this electrophilic reactivity is
often likely to be responsible for the bioactivity of 15-d-
PGJ , all of the deoxy-J -IsoPs are expected to have similar
lution of inflammation (50–52). 15-d-PGJ has, in fact,
been reported to be present in significant amounts in
human atherosclerotic lesions and to reside in foam cells
2
(50). 15-d-PGJ has also been detected in the inflamma-
2
2
2
bioactivities related to covalent interaction with intracellu-
tory exudates from carrageenan-induced pleurisy in rats,
using enzyme immunoassay (51), and it was detected by
LC/MS in the cell-free inflammatory exudates of mice
during resolving peritonitis (52). Additionally, stimulation
of selenium-supplemented murine macrophages with li-
popolysaccharide was shown to result in a time-dependent
lar proteins. In this regard, the deoxy-J -IsoPs represent a
2
new class of lipid peroxidation products that are predicted
to have more potent bioactivity than the previously identi-
fied cyclopentenone (A /J ) IsoPs, due to the presence of
2
2
two reactive electrophilic carbons. These compounds may
be important lipid mediators involved in the pathophysiol-
ogy of oxidant stress.
increase in 15-d-PGJ formation, quantified in cell lysates
2
by enzyme immunoassay (57). This suggests that 15-d-PGJ2
The extent to which both 15-d-PGJ and other deoxy-J -
and deoxy-J -IsoPs may be produced in vivo in settings of
2
2
2
IsoPs are produced in vivo is biologically relevant because
5-d-PGJ has been shown to mediate a wide range of bio-
inflammation in which COX activity is increased as well as
under conditions of oxidant stress.
1
2
logical activities, including modulating inflammatory and
apoptotic processes (35–41). Unlike other PGs, which
elicit their biological responses by binding specific G-pro-
Further support for the free radical-catalyzed genera-
tion of 15-d-PGJ and other deoxy-J -IsoPs in vivo, indepen-
2
2
dent of COX enzymes, was provided by the finding that
these compounds are formed in situ esterified in mem-
brane phospholipids in rat liver after induction of oxidant
injury by CCl . Hydrolysis of deoxy-J -IsoPs by bee venom
tein-coupled receptors (GPCRs), 15-d-PGJ exerts its bio-
2
logical effects through interaction with key intracellular
targets. Some of its activity may relate to the observation
4
2
that 15-d-PGJ activates the nuclear receptor PPAR-␥ (9).
PLA permitted detection and quantification of these
2
2
Activation of PPAR-␥ by 15-d-PGJ has been recently shown
compounds by LC/MS/MS. Free 15-d-PGJ , which would
2
2
1
22
Journal of Lipid Research Volume 52, 2011

be generated through the COX pathway, was not detect-
able in the liver of control rats and rats treated with CCl4.
9. Forman, B. M., P. Tontonoz, C. Jasmine, R. P. Brun, B. M.
12,14
Spiegelman, and R. M. Evans. 1995. 15-deoxy-⌬
-prostaglandin
J2 is a ligand for the adipocyte determination factor PPAR␥. Cell.
83: 803–812.
This may be due to the rapid reactivity of 15-d-PGJ . Previ-
2
ous studies have indicated that cyclopentenone eico-
sanoids rapidly undergo conjugation with GSH in vitro
and in vivo (26–30). Brunoldi et al. (26) reported that
10. Halliwell, B., and J. M. Gutteridge. 1990. Role of free radicals and
catalytic metal ions in human disease: an overview. Methods Enzymol.
1
86: 1–85.
1
1
1
1
1. Southorn, P. A., and G. Powis. 1988. Free radicals in medicine. II.
Involvement in human disease. Mayo Clin. Proc. 63: 390–408.
2. Ames, B. N. 1983. Dietary carcinogens and anticarcinogens. Oxygen
radicals and degenerative diseases. Science. 221: 1256–1264.
3. Harman, D. 1981. The aging process. Proc. Natl. Acad. Sci. U S A. 78:
7124–7128.
HepG2 cells convert 15-d-PGJ to a GSH conjugate in
2
which the carbonyl on the prostane ring is reduced to a
hydroxyl, and the GSH portion of the molecule is subse-
quently hydrolyzed to a cysteine conjugate. Additionally,
Milne et al. (58) showed that the cyclopentone eicosanoid
4. Morrow, J. D., K. E. Hill, R. F. Burk, T. M. Nammour, K. F. Badr,
and L. J. Roberts II. 1990. A series of prostaglandin F -like com-
2
1
5-A -IsoP is metabolized primarily in vivo by conjugation
2t
pounds are produced in vivo in humans by a non-cyclooxygenase,
free radical-catalyzed mechanism. Proc. Natl. Acad. Sci. U S A. 87:
9383–9387.
5. Stafforini, D. M., J. R. Sheller, T. S. Blackwell, A. Sapirstein, F.
E. Yull, T. M. McIntyre, J. V. Bonventre, S. M. Prescott, and L. J.
with GSH and that the compound is excreted into the
urine as the N-acetyl cysteine sulfoxide conjugate of 15-A -
2
t
1
IsoP in which the carbonyl on the prostane ring of the IsoP
is reduced to a hydroxyl. Preliminary studies suggest that,
Roberts II. 2006. Release of free F -isoprostanes from esterified
2
like other cyclopentenone eicosanoids, 15-d-PGJ is rap-
phospholipids is catalyzed by intracellular and plasma platelet-
activating factor acetylhydrolases. J. Biol. Chem. 281: 4616–4623.
6. Morrow, J. D., J. A. Awad, H. J. Boss, I. A. Blair, and L. J. Roberts II.
2
idly metabolized in vivo via conjugation with GSH and ex-
creted into the urine as a metabolized GSH adduct (data
not shown). Further studies are required to identify the
1
1
1
1
1
992. Non-cyclooxygenase-derived prostanoids (F -isoprostanes)
2
are formed in situ on phospholipids. Proc. Natl. Acad. Sci. USA. 89:
0721–10725.
1
major urinary metabolite of 15-d-PGJ to develop a bio-
2
7. Pryor, W. A., J. P. Stanley, and E. Blair. 1976. Autoxidation of
polyunsaturated fatty acids: II. A suggested mechanism for the for-
mation of TBA-reactive materials from prostaglandin-like endoper-
oxides. Lipids. 11: 370–379.
8. Morrow, J. D., T. M. Harris, and L. J. Roberts II. 1990. Noncy-
clooxygenase oxidative formation of a series of novel prostaglan-
dins: analytical ramifications for measurement of eicosanoids.
Anal. Biochem. 184: 1–10.
marker of its systemic production in humans. These studies
are critical to determine to what extent 15-d-PGJ is gener-
2
ated in vivo in humans and elucidate the mechanisms that
regulate its production.
In summary, we report the formation of cyclopentenone
eicosanoids 15-d-PGJ and deoxy-J -IsoPs in vivo via free
2
2
9. Morrow, J. D., T. A. Minton, C. R. Mukundan, M. D. Campbell, W.
E. Zachert, V. C. Daniel, K. F. Badr, I. K. Blair, and L. J. Roberts II.
1994. Free radical-induced generation of isoprostanes in vivo: evi-
dence for the formation of D-ring and E-ring isoprostanes. J. Biol.
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0. Taber, D. F., J. D. Morrow, and L. J. Roberts II. 1997. A nomencla-
ture system for the isoprostanes. Prostaglandins. 53: 63–67.
radical-catalyzed lipid peroxidation, independent of COX.
Deoxy-J -IsoPs represent a new class of reactive lipid per-
2
oxidation products that may exert unique biological ac-
tions relevant to the pathobiology of oxidant stress. This
study provides the rational basis with which to examine
2
the extent to which deoxy-J -IsoPs are generated in vivo in
21. Hirata, Y., H. Hayashi, S. Ito, Y. Kikawa, M. Ishibashin, M. Sudo,
H. Miyazaki, M. Fukushima, S. Narumiya, and O. Hayaishi. 1988.
2
humans. Additionally, future studies aimed at exploring
9
12
Occurrence of 9-deoxy-⌬ ,⌬ -13,14-dihydroprostaglandin D in
2
the biological activities of deoxy-J -IsoPs will likely yield in-
2
human urine. J. Biol. Chem. 263: 16619–16625.
sights into the pathophysiological consequences of their
formation in settings of oxidant stress.
2
2. Bell-Parikh, L. C., T. Ide, J. A. Lawson, P. McNamara, M. Reilly, and
1
2,14
G. A. FitzGerald. 2003. Biosynthesis of 15-deoxy-⌬
ligation of PPAR␥. J. Clin. Invest. 112: 945–955.
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2
2
3. Attallah, A., W. Payakkapan, J. Lee, A. Carr, and E. Brazelton. 1974.
PGA: fact, not artifact. Prostaglandins. 5: 69–71.
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