Formation of Reactive Cyclopentenone Compounds in
Vivo as Products of the Isoprostane Pathway*
Yan
Chen,
Jason D.
Morrow, and
L. Jackson
Roberts II
From the Departments of Pharmacology and Medicine, Vanderbilt
University, Nashville, Tennessee 37232-6602
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ABSTRACT |
Cyclopentenone prostaglandins
A2 and J2 are reactive compounds that
possess unique biological activities. However, the extent to which they
are formed in vivo remains unclear. In this study, we
explored whether D2/E2-isoprostanes undergo
dehydration in vivo to form
A2/J2-isoprostanes. Oxidation of arachidonic
acid in vitro generated a series of compounds that were
confirmed to be A2/J2-isoprostanes by mass
spectrometric analyses. A2/J2-isoprostanes were
detected in vivo esterified to lipids in livers from normal rats at a level of 5.1 ± 2.3 ng/g, and levels increased
dramatically by a mean of 24-fold following administration of
CCl4. An A2-isoprostane, 15-A2t-isoprostane, was obtained and found to readily
undergo Michael addition with glutathione and to adduct covalently to protein. A2/J2-isoprostanes could not be
detected in the circulation, even following CCl4
administration, which we hypothesized might be explained by rapid
formation of adducts. This was supported by finding that essentially
all the radioactivity excreted into the urine following infusion of
radiolabeled 15-A2t-isoprostane into a human volunteer was
in the form of a polar conjugate(s). These data identify a new class of
reactive compounds that are produced in vivo as products of
the isoprostane pathway that can exert biological effects relevant to
the pathobiology of oxidant injury.
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INTRODUCTION |
Cyclopentenone (CP)1
prostaglandins (PG) of the A and J series have been shown to be
produced in vitro by dehydration of the cyclopentane ring of
PGE2 and PGD2, respectively. These compounds have attracted considerable attention because they exert unique biological actions. CP-PGs are actively incorporated into cells and
accumulate in the nucleus (1, 2). They have been shown to inhibit
cellular proliferation with a G1 cell cycle arrest and to
induce differentiation, an effect that may be related to their ability
to modulate a variety of growth-related and stress-induced genes
(3-5). These cytostatic effects can be reversible, but higher
concentrations are cytotoxic and induce apoptosis (4, 6, 7).
Interestingly, at very low concentrations, PGA was found to stimulate
cellular proliferation (8). CP-PGs can also activate nuclear peroxisome
proliferator-activated receptor-
and suppress macrophage activation
and inflammatory responses (9-11). Furthermore, CP-PGs exhibit
antiviral activity (12). The common feature in these compounds is the
presence of a reactive
,
-unsaturated carbonyl group, which is
very susceptible to nucleophilic addition reactions and seems to be
essential for many of their biological effects (13-15).
Although the biological effects exerted by CP-PGs have been studied in
some detail, the extent to which they are formed in vivo has
been the subject of continuing controversy for over 2 decades (16-18).
Fueling this controversy has always been the uncertainty as to what
extent dehydration of PGE2 and PGD2 ex
vivo during sample processing contributes to the amount of
PGA2 and PGJ2 detected. Recently,
12-PGJ2 was definitively identified in human
urine by Hayaishi and co-workers (19). However, the amounts in urine
from males were >2-fold higher than the amounts in urine from females.
This is difficult to reconcile with the evidence suggesting that there is no sexual difference in the amount of PGD2 produced
in vivo in humans (20). Convincing evidence was presented
that the
12-PGJ2 detected in urine unlikely
arose as a result of dehydration of urinary PGD2 ex
vivo during sample processing. However, it is difficult to know to
what extent PGD2 may undergo dehydration in the
genitourinary tract prior to voiding. This is of particular interest
since the same authors recently reported that high levels of PGD
synthase are present in human male reproductive organs and that seminal
plasma greatly facilitates dehydration of PGD2 (21).
Furthermore, they also recently reported that the level of PGD synthase
in male urine is approximately twice that found in female urine (22).
Taken together, these findings suggest that at least some of the
12-PGJ2 detected in urine may have arisen
from dehydration of PGD2 in the genitourinary tract and may
explain the higher levels of
12-PGJ2 in
urine from males. This does not confute the occurrence of
12-PGJ2 in human urine, but only raises the
question of its origin, that being whether it arose from systemic
sources or from local production in the genitourinary tract. Therefore,
it still remains unclear whether CP-PGs are ubiquitously produced
throughout the body.
Free radicals have been increasingly implicated in the pathogenesis of
a wide spectrum of human disease processes (23, 24). Previously, we
reported the discovery of PG-like compounds, now termed isoprostanes
(IsoPs), that are produced in vivo nonenzymatically as
products of free radical-induced peroxidation of arachidonoyl lipids
(25, 26). Our initial report described the formation of
PGF2-like compounds (F2-IsoPs). Subsequently,
we reported the finding that PGD2- and
PGE2-like compounds (D2/E2-IsoPs)
are also produced in abundance in vivo (27). In the studies
reported herein, we explored whether
D2/E2-IsoPs undergo dehydration in vivo to produce CP-IsoPs (A2/J2-IsoPs).
Our interest in this stems from the fact that such compounds may be
identified as a new class of reactive compounds that are formed as
products of free radical-induced lipid peroxidation that exert unique
biological actions relevant to the pathobiology of oxidative stress.
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EXPERIMENTAL PROCEDURES |
Conversion of 15-E2t-IsoP to
15-A2t-IsoP--
8-Iso-[3H]PGA2,
termed 15-A2t-IsoP according to the approved nomenclature
for IsoPs (28), was obtained by treatment of
15-E2t-[3H]IsoP (8-iso-PGE2)
(Cayman Chemical Co., Inc., Ann Arbor, MI) with 0.1 N HCl
(29, 30), purified by normal phase HPLC, and analyzed by NMR for
structural confirmation.
Analysis of
A2/J2-IsoPs--
A2/J2-IsoPs
were analyzed by GC/NICI/MS using a modification of methods described
for the analysis of other IsoPs (31). Briefly, 10 ng of
[2H4]PGA2 was added to samples as
an internal standard, after which compounds were partially purified
using C18 and Silica Sep-Pak cartridges (Waters),
methoximated, and converted to pentafluorobenzyl (PFB) esters.
Compounds were then purified by chromatography on silica 60ALK6D TLC
plates (Whatman) using a solvent system of hexane/acetone (70:30, v/v).
Compounds migrating in the region from 0.5 cm below the PFB ester of
PGJ2 to 1 cm above the PFB ester of PGA2 were
scraped, extracted with ethyl acetate, converted to a trimethylsilyl
(TMS) ether derivative, and then analyzed by GC/NICI/MS. The major ions
generated in the NICI mass spectra of the PFB ester,
O-methyloxime, TMS ether derivative of
A2/J2-IsoPs and
[2H4]PGA2 are the carboxylate
anions at m/z 434 and m/z
438, respectively. Quantitation was performed by integration of the
peak areas.
Analysis of A2/J2-IsoPs as a
Piperidyl-enol-trimethylsilyl Ether Derivative--
Treatment of PGA
with N,O-bis(trimethylsilyl)trifluoroacetamide
(BSTFA) and piperidine has been shown to convert it to a
piperidyl-enol-TMS ether derivative, which is specific for
A-ring prostanoids (32). Thus, we analyzed for the formation of this
derivative with CP-IsoPs. PFB esters of putative CP-IsoPs were treated
with a 1:1 mixture of BSTFA/piperidine for 1 h at 60 °C and
analyzed by GC/NICI/MS monitoring of the carboxylate anions at
m/z 562 for the IsoPs and
m/z 566 for
[2H4]PGA2.
Analysis of A2/J2-IsoPs by GC/Electron
Ionization/MS--
Putative A2/J2-IsoPs
generated during oxidation of arachidonic acid in vitro were
subjected to purification using two HPLC systems: (a)
normal-phase HPLC on a 5-µm Econosil SI column using an isocratic
solvent system of hexane/isopropyl alcohol/acetic acid (97:3:0.1,
v/v/v) and (b) reversed-phase HPLC on a 5-µm Econosil C18 column using an isocratic solvent system of
acetonitrile/water/acetic acid (38:62:0.1, v/v/v).
15-A2t-[3H]IsoP was added prior to HPLC
purification, and compounds that coeluted with
15-A2t-[3H]IsoP were then converted to a PFB
ester, O-methyloxime, TMS ether derivative and analyzed by
GC/electron ionization/MS.
Analysis of D2/E2-IsoPs by
GC/NICI/MS--
Analysis of D2/E2-IsoPs by
GC/NICI/MS was performed as described (27). The amounts of
D2/E2-IsoPs reported in this study differ from
those in our previous reports (27). In our previous reports,
quantification was based on the intensity of a single prominent peak,
whereas in this study, the integrated area under all peaks was used for quantification.
Oxidation of Arachidonic Acid in Vitro--
Non-esterified
arachidonic acid was dissolved in ethanol and then oxidized in 50 mM Tris buffer for 45 min using an iron/ADP/ascorbate oxidizing system as described (33).
1-Palmitoyl-2-arachidonoyl-phosphatidylcholine was prepared and
oxidized in an identical fashion as non-esterified arachidonic acid at
a concentration equivalent to 1 mg/ml arachidonic acid. After
oxidation, the phospholipid was extracted and hydrolyzed as described
below for liver phospholipids.
In Vivo Model of Free Radical-induced Liver Injury--
Free
radical-induced lipid peroxidation in liver was induced in rats by
administration of CCl4 (25). Two hours after treatment, animals were killed. Livers were then removed, snap-frozen in liquid
N2, and either processed immediately or stored at
-70 °C.
Extraction, Purification, and Hydrolysis of
Phospholipids--
Lipids from livers of CCl4-treated and
control rats were extracted as described (31). The lipid extracts were
then hydrolyzed enzymatically using Apis mellifera bee venom
phospholipase A2 (27).
Conjugation of 15-A2t-IsoP and PGA2 with
GSH in Vitro--
Radiolabeled 15-A2t-IsoP and
PGA2 were incubated at 37 °C with GSH in phosphate
buffer (pH 6.5) in the presence of 19 units/ml glutathione
S-transferase (Sigma). The molar ratio of the prostanoids to
GSH was 1:10. At the designated time points, aliquots were removed,
acidified immediately to pH 3, and extracted twice with 2 volumes of
methylene chloride. The formation of a GSH conjugate was assessed by
determining the percent of radioactivity that did not extract into
organic solvent (14). We predetermined that 93 ± 4% (mean ± S.E.) of PGA extracts into methylene chloride from buffer solutions
at pH 3. Control incubations were carried out in the absence of GSH and enzyme.
Formation of 15-A2t-IsoP and PGA2
Covalent Adducts with Albumin--
Radiolabeled PGA2 and
15-A2t-IsoP were incubated at 37 °C with 20 mg/ml human
serum albumin in phosphate buffer (pH 7.4). The molar ratio of
prostanoids to albumin was 1:20. At the designated time points,
aliquots were withdrawn, and 10 volumes of cold ethanol were
immediately added to precipitate the albumin. The formation of covalent
albumin adducts was assessed by determining the percent of
radioactivity present in the albumin precipitate after centrifugation. Control incubations were carried out in the absence of albumin.
Formation of Polar Conjugates of 15-A2t-IsoP in
Humans--
After informed consent was obtained, a small tracer
quantity (1 µCi) of 15-A2t-[3H]IsoP (150 Ci/mmol) was dissolved in 200 µl of ethanol, diluted to 10 ml with
sterile saline, and infused over 10 min into the antecubital vein of a
normal male volunteer. Individual voided urine specimens were collected
over a period of 10 h and assayed for radioactivity. The formation
of polar conjugates was assessed by determining the percent of
radioactivity in a 1-ml aliquot of urine that did not extract into 2 volumes of methylene chloride at pH 3.
 |
RESULTS |
Initially, we explored whether A2/J2-IsoPs
are formed during oxidation of arachidonic acid in vitro
(Fig. 1). In the upper m/z 434 chromatogram are multiple peaks with a
similar retention time as the m/z 438 peaks
representing the syn- and
anti-O-methyloxime isomers of
[2H4]PGA2 (lower chromatogram),
consistent with the formation of CP-IsoPs. The amount of the compounds
formed was 529 ± 135 ng/mg of arachidonic acid (mean ± S.E., n = 4).

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Fig. 1.
Selected ion current chromatograms from the
analysis of the formation of A2/J2-IsoPs during
oxidation of arachidonic acid in vitro. The peaks in the
m/z 438 ion current chromatogram represent the
syn- and anti-O-methyloxime isomers of
the [2H4]PGA2 internal standard.
In the m/z 434 chromatogram are a series of peaks
consistent with the presence of A2/J2-IsoPs.
The summed total amount of the putative
A2/J2-IsoPs formed was 529 ng/mg of arachidonic
acid.
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Additional analyses further supported the identity of these compounds
as A2/J2-IsoPs. First, no
m/z 433 peaks were present, indicating that the
m/z 434 peaks were not natural isotope peaks of
compounds generating an ion less than m/z 434. When analyzed as [2H9]TMS ether and
O-[2H3]methyloxime derivatives,
all of the original m/z 434 peaks disappeared,
and an identical pattern of new peaks appeared 9 and 3 Da higher,
respectively (data not shown), indicating that all of the compounds had
one hydroxyl and one carbonyl group. When analyzed following catalytic
hydrogenation, there was a disappearance of the
m/z 434 peaks and the appearance of new intense
peaks 6 Da higher at m/z 440 (Fig.
2). No peaks were detected at
m/z 436, 438, or 442. This indicated that the
compounds contained three double bonds. In addition, analysis following
treatment with BSTFA/piperidine resulted in the formation of a
piperidyl-enol-TMS ether derivative (Fig.
3). The amount of
A2/J2-IsoPs analyzed as this derivative was
calculated to be 118 ± 32 ng/mg of arachidonic acid (mean ± S.E., n = 4), which is less than the amount formed when
analyzed as an O-methyloxime, TMS ether derivative. This
discrepancy can be explained by the fact that we have found that,
whereas treatment of PGA2 with BSTFA/piperidine efficiently
converts it to the piperidyl-enol-TMS ether derivative, only
trivial amounts of this derivative are formed with PGJ2. By
analogy, therefore, only a portion of the mixture of
A2/J2-IsoPs would be expected to form this
derivative.

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Fig. 2.
Analysis of the putative
A2/J2-IsoPs formed during oxidation of
arachidonic acid in vitro prior to and after catalytic
hydrogenation. A, analysis of compounds prior to
hydrogenation. The peaks in the m/z 434 ion
current chromatogram represent putative
A2/J2-IsoPs, and the peaks in the
m/z 438 chromatogram represent the
[2H4]PGA2 internal standard. No
compounds were detected 6 Da above m/z 434 at
m/z 440 prior to hydrogenation (not shown).
B, analysis of compounds following hydrogenation. Both the
internal standard and the m/z 434 peaks in
A have shifted upwards 6 Da following hydrogenation,
indicating the presence of three double bonds.
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Fig. 3.
Analysis of
A2/J2-IsoPs generated during oxidation of
arachidonic acid in vitro as a PFB ester,
piperidyl-enol-TMS ether derivative. The
peaks in the m/z 566 chromatogram represent the
[2H4]PGA2 internal standard. In
the m/z 562 chromatogram are a series of peaks
consistent with the formation of a piperidyl-enol-TMS ether
derivative of CP-IsoPs.
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Further evidence for the identity of these compounds as CP-IsoPs was
obtained by analyzing the compounds by electron ionization/MS to obtain
complete mass spectra. Compounds were analyzed as a PFB ester,
O-methyloxime, TMS ether derivative. One of the mass spectra
obtained is shown in Fig. 4. As noted, a
molecular ion is present at m/z 615. Other
prominent ions are present at m/z 600 (M
15, loss of ·CH3), m/z
584 (M
31, loss of ·OCH3),
m/z 544 (M
71, loss of
·CH2(CH2)3CH3),
m/z 525 (M
90, loss of Me3SiOH), m/z 494 (M
(90 + 31)), m/z 308 (M
307, loss of
·CH2 CH=CH(CH2)3COOC6F5),
m/z 277 (M
(307 + 31)),
m/z 218 (M
(307 + 90)),
m/z 199 (+CH=CH-CH(Me3SiOH)(CH2)4CH3),
m/z 181 (base)
(+CH2C6F5), and
m/z 173 (Me3SiO+=CH(CH2)4CH3).
These ions or analogous ions and losses of other derivatives are also
intense ions in the mass spectra of CP-PGs (34),2 indicating that this
was a mass spectrum of a 15-series CP-IsoP in which the side chain
hydroxyl is located at C-15. Although the structure depicted in Fig. 4
is an A-ring IsoP, ions are not present that would allow a
differentiation between an A-ring and a J-ring compound.

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Fig. 4.
Representative electron ionization mass
spectrum obtained from the analysis of
A2/J2-IsoPs generated from oxidation of
arachidonic acid in vitro as a PFB ester,
O-methyloxime, TMS ether derivative. See
"Results" for interpretation of the mass spectrum.
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We then explored whether CP-IsoPs are also formed in vivo.
Previously, we showed that IsoPs are initially formed in
situ esterified in tissue phospholipids (26). Therefore, we
assayed for the presence of A2/J2-IsoPs
esterified in livers from rats that had been treated with
CCl4 to induce lipid peroxidation (Fig.
5A). Again, a series of
m/z 434 peaks were present similar to what was
found following oxidation of arachidonic acid in vitro,
although the pattern of the peaks differed somewhat. This may be
explained by our observation that there appears to be a steric
influence of phospholipids on the formation of different IsoP isomers.
In support of this notion, the pattern of peaks present following oxidation of arachidonoylphosphatidylcholine in vitro was
found to be very similar to the pattern of peaks detected in esterified lipids in liver (Fig. 5B).

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Fig. 5.
A, analysis of
A2/J2-IsoPs formed in vivo
esterified in lipids in the liver of a rat treated with
CCl4. The peaks in the m/z 441 ion
current chromatogram represent the
[2H4]PGA2 internal standard. In
the m/z 434 ion current chromatogram are a series
of peaks consistent with the presence of
A2/J2-IsoPs. B, analysis of
A2/J2-IsoPs following oxidation of
arachidonoyl-phosphatidylcholine in vitro. In the
m/z 434 chromatogram is a series of peaks
consistent with the presence of A2/J2-IsoPs in
a pattern that is very similar to the pattern of peaks detected in rat
liver.
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Further evidence for the identity of these compounds generated in
vivo as A2/J2-IsoPs was obtained utilizing
similar approaches used for the structural identification of the
compounds formed in vitro. Analysis as a
[2H9]TMS ether derivative, an
O-[2H3]methyloxime derivative, and
following catalytic hydrogenation indicated that all of the compounds
had one hydroxyl group, one carbonyl, and three double bonds (data not
shown). Furthermore, the compounds formed a
piperidyl-enol-TMS ether derivative (Fig. 6), and the pattern of peaks was almost
identical to that formed by the CP-IsoPs generated in vitro
(Fig. 3), although the relative abundance of individual peaks differed
somewhat.

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Fig. 6.
Analysis of CP-IsoPs esterified in the liver
of a rat treated with CCl4 as a PFB ester,
piperidyl-enol-TMS ether derivative. The
peaks in the m/z 562 ion current chromatogram
represent CP-IsoPs, and the m/z 566 peaks
represent the [2H4]PGA2 internal
standard.
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We then compared the relative amounts of
A2/J2-IsoPs and
D2/E2-IsoPs present in livers from both normal
and CCl4-treated rats to determine the extent to which
D2/E2-IsoPs undergo dehydration in
vivo (Fig. 7). Of interest was the
finding that A2/J2-IsoPs could be detected
esterified in the livers of normal rats at a level of 5.1 ng/g of
liver. Relative to the amounts of D2/E2-IsoPs measured, A2/J2-IsoPs were present at levels
indicating that the extent to which D2/E2-IsoPs
undergo dehydration in vivo is not inconsequential.
Following administration of CCl4, the levels of both
A2/J2-IsoPs and
D2/E2-IsoPs increased dramatically and to a
similar extent by a mean of 23.9- and 21.2-fold, respectively.

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Fig. 7.
Analysis of CP-IsoPs and
D2/E2-IsoPs esterified in livers from normal
rats and following administration of CCl4 to induce an
oxidant injury to the liver.
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We then carried out experiments to exclude the possibility that
dehydration of D2/E2-IsoPs ex vivo
during sample processing contributed significantly to the levels
measured in liver. That this was not the case was supported by the
finding that <1% of either PGE2 or PGD2 that
was added to a lipid extract of liver and then processed and analyzed
had undergone dehydration to form PGA2 and
PGJ2, respectively.
Our major interest in the possibility that CP-IsoPs may be formed
in vivo is because these compounds should be reactive
molecules that are susceptible to nucleophilic addition reactions.
Therefore, we compared the ability of one of the CP-IsoPs,
15-A2t-IsoP, and PGA2 to conjugate with GSH and
adduct to protein, using albumin as a model. We recently showed that
one of the E2-IsoPs that is produced in vivo is
15-E2t-IsoP (25). Its dehydration product, 15-A2t-IsoP should therefore also be one of the CP-IsoPs
that is produced in vivo. We initially determined the time
course of conjugation of GSH with radiolabeled 15-A2t-IsoP
and PGA2 in the presence of glutathione
S-transferase. Formation of GSH conjugates was assessed by
determining the percent of radioactivity that did not extract into
methylene chloride at pH 3. Approximately 70% of
15-A2t-IsoP had conjugated with GSH within 2 min, and the conjugation was complete by 8 min (Fig.
8). The time course for the conjugation
of PGA2 with GSH was found to be essentially identical. In
the absence of glutathione S-transferase, no appreciable
conjugation occurred with either compound over the same time period
(data not shown). Similarly, the time course for the formation of
covalent adducts with albumin was essentially identical for both
PGA2 and 15-A2t-IsoP (Fig.
9).

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Fig. 8.
Time course of GSH-catalyzed conjugation of
15-A2t-IsoP and PGA2 with GSH. Formation
of polar GSH conjugates was monitored over time and is expressed as the
percent of total radioactivity that did not extract into methylene
chloride.
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Fig. 9.
Time course of covalent adduction of
15-A2t-IsoP and PGA2 with albumin.
Formation of the adducts was monitored over time and is expressed as
the percent of total radioactivity present in the protein pellet
following precipitation with cold ethanol.
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Following administration of CCl4 to rats, plasma
concentrations of non-esterified F2-IsoPs and
D2/E2-IsoPs increase to very high levels (25,
27). However, we unable to detect CP-IsoPs in the circulation of rats
following administration of CCl4 in the experiments
described above. We hypothesized that this may be due to rapid
formation of polar conjugates, e.g. with GSH, following the
hydrolysis of the compounds from phospholipids. To test this
hypothesis, we infused a tracer quantity of radiolabeled 15-A2t-IsoP into a normal human volunteer and determined
the percent of radioactivity excreted into the urine that did not
extract into methylene chloride. Approximately 95% of the
radioactivity recovered in the urine was excreted during the first
4 h following the infusion, representing ~30% of the total
amount of radioactivity infused. Only ~5% of the radioactivity
present in the urine was recovered in the organic extract, suggesting
that all or almost all of the compounds were present in the form of a
polar conjugate(s).
 |
DISCUSSION |
We report the finding that PGA2- and
PGJ2-like compounds are formed in vivo as
products of the IsoP pathway. Important and interesting was the finding
that these compounds not only were detected in abundant quantities
esterified in rat livers following induction of an oxidant injury, but
were also present in readily detectable quantities in the livers of
normal rats.
The discovery of the formation of CP-IsoPs opens up numerous new
avenues for scientific inquiry. The reactive nature of these compounds
conferred by the
,
-unsaturated carbonyl moiety that characterizes
these molecules provides a basis for hypotheses regarding their
potential role in the pathogenesis of oxidant injury. As mentioned
previously, CP prostanoids exert unique biological effects. They
inhibit cellular proliferation via their ability to modulate a variety
of growth-related and stress-induced genes, induce apoptotic cell death
at higher concentrations, and activate peroxisome
proliferator-activated receptor-
(3-11). The reactive
,
-unsaturated carbonyl seems essential for many of these
biological actions (15). In this regard, one might anticipate that all of the CP-IsoPs might exert similar biological effects, lessening the
potential importance of elucidating the precise structures of
individual compounds in the mixture. As shown in Fig. 8,
PGA2 and 15-A2t-IsoP covalently adduct to
protein. Interestingly, however, in intact cells, PGA2
seems to preferentially bind to particular proteins, the functions of
which remain to be elucidated (35). As shown in Fig. 7,
PGA2 and 15-A2t-IsoP also rapidly undergo glutathione S-transferase-catalyzed conjugation with GSH. We
have previously shown that
12-PGJ2 also
rapidly conjugates with GSH, but does require catalysis by glutathione
S-transferase (14). However, we (36) and others (37) have
shown that conjugation with GSH acts to prevent the ability of CP-PGs
to inhibit cellular proliferation and to induce apoptosis. Therefore,
much remains to be known about the underlying molecular mechanisms that
mediate the biological effects of CP prostanoids. Furthermore, the
breadth of our understanding of the biological actions of CP
prostanoids is probably far from complete. For example, virtually
nothing is known about the biological consequences of the adduction of
these molecules with DNA in regards to their potential mutagenicity.
Dehydration of PGE2 and PGD2 occurs in
physiologic buffers, but has also been shown to be catalyzed by plasma
and albumin and more recently also by human semen (21, 28, 38, 39). Plasma components could be involved in promoting dehydration of E-ring
and D-ring IsoPs following their release from tissue phospholipids into
the circulation. However, this study, we identified the presence of
CP-IsoPs esterified in tissue membrane lipids. Whether there are also
factors in cellular membranes that promote the dehydration of
D2/E2-IsoPs remains to be explored.
Nonetheless, identification of factors that may promote this
dehydration may reveal potential avenues for intervention to modulate
the formation of CP-IsoPs in vivo.
Our observation that 15-A2t-IsoP undergoes extensive
conjugation in vivo in humans is consistent with our
previous findings that
12-PGJ2 also
undergoes extensive conjugation in rats (14). These observations have
potentially very important implications. This may explain the
difficulty in the past in demonstrating the formation of CP-PGs
in vivo because efforts were focused on detecting these compounds in free form. Even if detected in free form in small amounts,
e.g. in urine as reported by Hirata et. al (19),
this could potentially lead to the erroneous conclusion that the
amounts formed in vivo may not be biologically relevant.
However, quantification of the conjugated form(s) of CP-IsoPs in plasma
and/or urine may provide an approach that more accurately assesses the
magnitude of endogenous production of both CP-PGs and CP-IsoPs. In the
case of CP-IsoPs, it is possible to assess their formation esterified in tissues of interest, but this is not amenable to human
investigation. More important, as well, measurement of a conjugate of
CP-PGs and CP-IsoPs would completely eliminate the confounding problem of artifactual formation ex vivo of what is being measured.
This clearly provides the impetus for future studies aimed at
elucidating the nature of these conjugates.
In summary, we have reported the discovery of a new class of reactive
products of lipid peroxidation that are formed in vivo via
the IsoP pathway. This provides a rational basis to explore in depth
the biological activities of these novel molecules that may provide new
insights into the biological consequences of their formation as it
relates to the pathobiology of oxidant injury.
 |
ACKNOWLEDGEMENT |
We extend our appreciation to Dr. Thomas
Harris for valuable assistance in the NMR analysis of
15-A2t-IsoP.
 |
Addendum |
Since submission of the manuscript, we carried out a
study to determine if 15-A2t-IsoP (8-iso-PGA2)
is one of the CP-IsoPs that is produced in vivo. We found
that a single compound isolated from the livers of
CCl4-treated rats coeluted with radiolabeled 15-A2t-IsoP through four high resolving HPLC purification
procedures, indicating that 15-A2t-IsoP is in fact one of
the CP-IsoPs formed in vivo. Furthermore, the data indicated
that the first two HPLC procedures eliminated all other CP-IsoPs. This
suggests that the mass spectrum of the compound shown in Fig. 4 that
coeluted with radiolabeled 15-A2t-IsoP through two HPLC
purification procedures is likely a mass spectrum of
15-A2t-IsoP.
 |
FOOTNOTES |
*
This work was supported by National Institutes of Health
Grants GM42057, GM15431, and DK48831.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed. Tel.: 615-343-1816;
Fax: 615-322-4707; E-mail: jack.roberts{at}mcmail.vanderbilt.edu.
2
Y. Chen, W. E. Zackert, L. J. Roberts II, and J. D. Morow unpublished data.
 |
ABBREVIATIONS |
The abbreviations used are:
CP, cyclopentenone;
PG, prostaglandin;
IsoP, isoprostane;
HPLC, high pressure liquid
chromatography;
GC, gas chromatography;
NICI, negative ion chemical
ionization;
MS, mass spectrometry;
PFB, pentafluorobenzyl;
TMS, trimethylsilyl;
BSTFA, N,O-bis(trimethylsilyl)trifluoroacetamide.
 |
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