From the
Departments of Medicine,
Pharmacology,
¶Pediatrics, and Chemistry,
||Vanderbilt University, Nashville, Tennessee 37232
and the **College of Pharmacy, University of Kentucky,
Lexington, Kentucky 40536
Received for publication, April 16, 2003 , and in revised form, May 5, 2003.
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ABSTRACT |
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INTRODUCTION |
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We have previously reported that a series of PG-like compounds termed
isoprostanes (IsoPs) are formed in vivo from the free
radical-catalyzed peroxidation of arachidonate independent of COX
(6). Analogous to PGs, we have
determined that IsoPs contain E/D-, F-, and thromboxane-type prostane rings
(7). Although the structures of
these compounds are very similar to COX-derived PGs, an important distinction
between IsoPs and PGs is that IsoP bicycloendoperoxide intermediates contain
side chains that are predominantly (>90%) oriented cis in relation
to the prostane ring because the generation of these intermediates is favored
kinetically (4,
7,
8). Indeed, we have previously
reported that two IsoPs that are formed in abundance in vivo are
15-F2t-IsoP (8-iso-PGF2) and
15-E2t-IsoP (8-iso-PGE2), which are generated from the
endoperoxide intermediate 15-H2t-IsoP (8-iso-PGH2)
(Fig. 1B)
(9,
10). Although not reported, it
would also be predicted that 15-H2c-IsoP (12-iso-PGH2)
is formed in abundance and can rearrange to the analogous D-ring IsoP
15-D2c-IsoP (12-iso-PGD2)
(Fig. 1C).
In contrast to other types of prostanoids,
E2/D2-IsoPs are -hydroxyketone-containing
compounds that can undergo reversible keto-enol tautomerization under both
acidic and basic conditions, allowing rearrangement of the side chains that
are initially cis to the more stable trans-configuration.
That the trans-configuration is highly favored has been demonstrated
by the finding that, when PGE2 is subjected to conditions that
induce keto-enol tautomerism, <10% of the compound rearranges to the
cis-side chain isomer 15-E2t-IsoP
(11). In addition, attempts to
synthesize 15-D2c-IsoP have been unsuccessful because epimerization
at C-12 readily occurs during synthesis to yield PGD2
(12). Furthermore, facile
epimerization of a number of other PG-like compounds containing side chains
cis to the prostane ring has been reported
(13,
14).
In the course of studies to characterize various E/D-ring IsoPs formed in vitro and in vivo from the peroxidation of arachidonic acid, analysis of oxidation products by gas chromatography (GC)/mass spectrometry (MS) disclosed the generation of significant amounts of compounds that had retention times and molecular weights identical to those of PGE2 and PGD2. Because IsoPs are formed nonenzymatically, compounds generated by this pathway would be predicted to be racemic (6, 7). Using a variety of chromatographic and mass spectrometric approaches, we present evidence that compounds identical in all respects to COX-derived PGE2 and PGD2 and their respective enantiomers are formed in vitro and in vivo via the IsoP pathway. A proposed mechanism by which the formation of PGE2 and PGD2 occurs from 15-E2t-IsoP and 15-D2c-IsoP, respectively, via base-catalyzed isomerization is shown in Fig. 2 (A and B). Generation of PGE2 and PGD2 from 15-E2t-IsoP and 15-D2c-IsoP, respectively, would also be predicted to occur via acid catalysis (11).
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These findings strongly support the contention that a second pathway exists for the formation of bioactive PGs in vivo that is independent of COX. This finding is of potential physiological and pharmacological importance because it would be predicted that the generation of PGs via this mechanism would not be inhibited by aspirin or other COX inhibitors. For purposes of discussion hereafter, PGs possessing a structure identical to those generated by COX are referred to PGE2 and PGD2. Compounds that are enantiomeric to COX-derived PGs are referred to as ent-PGE2 and ent-PGD2. The racemic mixtures are termed rac-PGE2 and rac-PGD2 (Fig. 3).
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EXPERIMENTAL PROCEDURES |
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Epimerization of 15-E2t-IsoP in Phosphate Buffer15-E2t-IsoP (1 µM) was incubated in 50 mM KPO4 buffer, pH 7.4, for 024 h. Subsequently, fractions were analyzed for 15-E2t-IsoP, PGE2, 15-A2t-IsoP, and PGA2 by GC/MS and NMR as described (10, 16, 17).
Oxidation of Arachidonic AcidArachidonic acid was oxidized in vitro using an iron/ADP/ascorbate mixture as previously described (16, 17).
Isolation of E/D-ring IsoPs and PGs from Rodent and Human Tissue and UrineA mixture of E/D-ring IsoPs and PGs was isolated from the livers of Sprague-Dawley rats 2 h after intragastric administration of CCl4 (2 mg/kg) in corn oil (16, 17). The animals were anesthetized with pentobarbital (60 mg/kg) intraperitoneally and killed, and the livers were removed. Depending on the experiment, 14 g of tissue was immediately extracted to obtain a crude phospholipid extract containing IsoPs and PGs esterified in phospholipids. The lipid extract was then subjected to hydrolysis (30 min) in boronate buffer with A. mellifera venom containing phospholipase A2 as described (16, 17). In control experiments, complete hydrolysis of 2-[3H7]arachidonylphosphatidylcholine was effected during this incubation. In addition, these hydrolysis conditions resulted in <5% epimerization of 15-E2t-IsoP to PGE2. Subsequently, free IsoPs and PGs were extracted, partially purified using C18 and silica Sep-Pak cartridges, and subjected to HPLC. For selected experiments, liver tissue was also obtained from day 19 COX-1//COX-2/ mouse pups harvested in utero as described (18).
In some experiments, 24-h urine samples were collected from rats treated with CCl4 or from normal humans. Unesterified IsoPs and PGs were extracted using Sep-Pak columns as described (6).
HPLC Separation of Racemic E/D-ring PGs and
IsoPsDepending on the experiment, incubations of oxidized
arachidonic acid, partially purified tissue extracts, or urine samples were
analyzed for rac-PGE2, rac-PGD2,or
rac-15-E2t-IsoP
(9,
10). To the biological sample
was added 0.53 µCi of
[3H7]PGE2,
[3H7]PGD2, or
15-E2t-[3H6]IsoP. The mixture was then
subjected to four successive HPLC purification steps. To maximize purification
and resolution of each compound, we used HPLC procedures that yielded
relatively long retention volumes for each compound (
1540 ml), and
each solvent was run isocratically. In pilot experiments, it was also shown
that the three compounds readily separated from one another under the HPLC
conditions utilized. In addition, radiolabeled PGE2,
PGD2, and 15-E2t-IsoP separated to a significant extent
(12.5 ml) from unlabeled compounds due to the fact that the
radiolabeled compounds contained either six or seven tritium atoms. Thus, for
each HPLC step, fractions corresponding to those containing both labeled and
unlabeled PGs or IsoPs were collected and pooled for further purification. For
rac-PGE2 and rac-15-E2t-IsoP, the
first HPLC step was normal-phase using a Econosil SI column (25 cm x 4.6
mm, 5-µm particles; Alltech Associates Inc., Deerfield, IL). The solvent
system was 88:12:0.1 (v/v/v) hexane/isopropyl alcohol/acetic acid at a flow
rate of 1 ml/min. The second HPLC step was reversed-phase using an Econosil
C18 column (25 cm x 4.6 mm, 5 µm; Alltech Associates
Inc.). The solvent system was 30:70:0.1 (v/v/v) acetonitrile/water/acetic acid
at a flow rate of 1 ml/min. For the third and fourth HPLC steps, IsoPs or PGs
were converted to PFB esters and rechromatographed on normal- and
reversed-phase HPLC columns. A solvent system of 92:8 (v/v) hexane/isopropyl
alcohol was used for the third HPLC step, and 51:49 (v/v) acetonitrile/water
was used for the fourth HPLC step, both at a flow rate of 1 ml/min. For the
purification of rac-PGD2, the same columns were utilized,
but the solvent systems varied. For the first HPLC step, the solvent system
was 93:7:0.1 (v/v/v) hexane/isopropyl alcohol/acetic acid; the second HPLC
solvent system was 33:67:0.1 (v/v/v) acetonitrile/water/acetic acid; the third
HPLC solvent system was 95:5 (v/v) hexane/isopropyl alcohol; and the fourth
HPLC solvent system was 58:42 (v/v) acetonitrile/water.
Chiral HPLC Separation of rac-PGE2 and rac-PGD2Racemic PGs purified by the methods described above were subsequently subjected to chiral HPLC to separate enantiomers using a Chiralpak AD column (25 cm x 4.6 mm, 5 µm; Chiral Technologies, Exton, PA). To separate rac-PGE2, a solvent system of 93:7 (v/v) hexane/isopropyl alcohol was utilized; and for rac-PGD2, a solvent system of 95:5 (v/v) hexane/isopropyl alcohol was employed.
Analysis of PGs and IsoPs by GC/MSQuantification of E/D-ring PGs and IsoPs in partially purified biological extracts and throughout subsequent HPLC purification procedures was performed by analyzing aliquots by selected ion monitoring GC/negative ion chemical ionization MS using either [2H4]PGE2 or [2H4]PGD2 as an internal standard. Compounds were quantified as O-methyloxime, PFB ester, trimethylsilyl ether derivatives by monitoring the M-PFB (M 181) ions at m/z 524 for endogenous compounds and at m/z 528 for the deuterated standards (10, 19).
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RESULTS |
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Analysis of
E2/D2-IsoPs from the
Oxidation of Arachidonic Acid in Vitro and in
VivoFig.
5A shows the selected ion current chromatograms for
E2/D2-IsoPs obtained from the analysis of arachidonic
acid with iron/ADP/ascorbate for 2 h. Compounds were analyzed as
O-methyloxime, PFB ester, trimethylsilyl ether derivatives. In the
lower m/z 528 chromatogram are two peaks representing the
syn- and anti-O-methyloxime isomers of the
[2H4]PGE2 internal standard. In the upper
m/z 524 chromatogram are a series of peaks representing
various E2/D2-IsoPs. The peaks indicated by
asterisks represent compounds that co-chromatographed upon GC with
the O-methyloxime isomers of chemically synthesized PGE2.
In addition, the peaks denoted by plus signs co-chromatographed with
the O-methyloxime isomers of chemically pure PGD2. The
total E2/D2-IsoPs present were 1500 ng/g of
arachidonic acid. The materials designated by the peaks denoted by
asterisks and plus signs each represent
20% of the
total E2/D2-IsoPs in the mixture.
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In addition to the analysis of oxidized arachidonic acid in vitro,
Fig. 5B shows the
selected ion current chromatograms obtained from the hydrolysis of rat liver
phospholipids after administration of CCl4 to animals to induce
oxidant stress. As shown, a similar pattern of peaks was present as shown in
Fig. 5A. The analyses
in Fig. 5 (A and
B) were performed on separate days, accounting for
differences in GC retention time. Again, the peaks indicated by
asterisks represent compounds that co-chromatographed upon GC with
chemically synthesized PGE2, whereas those denoted by plus
signs co-chromatographed with PGD2. The total
E2/D2-IsoPs present in this sample were 400 ng/g of
liver tissue. The materials designated by the peaks denoted by the
asterisks and plus signs each represent
1520% of
the total E2/D2-IsoPs in the mixture. A very similar
pattern of peaks representing E2/D2-IsoPs was obtained
from the livers of both
COX-1//COX-2/
and control fetal mice without induction of oxidant stress, although total
E2/D2-IsoP levels were
510 ng/g of liver
tissue (data not shown). In addition, a pattern of peaks virtually identical
to those shown in the chromatograms in Fig.
5 was obtained after hydrolysis of rat liver phospholipids that
had been treated with methyloxime HCl prior to hydrolysis. These latter
findings suggest that epimerization of E2/D2-IsoPs
occurs while compounds are esterified in phospholipids. Taken together, these
data support the contention that significant amounts of compounds that coelute
upon GC with PGE2 and PGD2 are generated from the
peroxidation of arachidonic acid in vitro and in vivo.
Purification of Putative rac-PGE2 from Rat Liver Hydrolysates by HPLCWe subsequently sought to determine whether the compounds from rat liver hydrolysates that coeluted upon GC with PGE2 and PGD2 were, in fact, structurally identical to PGE2 and PGD2 and their respective enantiomers. If PGE2 and PGD2 are formed via the IsoP pathway, it would be predicted that they would be racemic mixtures because they would be formed from the epimerization of rac-15-E2t-IsoP and rac-15-D2c-IsoP, respectively (6, 7). Of note, enantiomers of PGE2 and PGD2 would not be expected to separate using standard non-chiral HPLC methods.
The formation of rac-PGE2 was assessed initially. For
these studies, 2000 ng of E2/D2-IsoPs from rat
liver containing 3 µCi of [3H7]PGE2 was
subjected to four successive HPLC purification steps. The first HPLC step was
normal-phase using a solvent system of 88:12:0.1 (v/v/v) hexane/isopropyl
alcohol/acetic acid. Aliquots of fractions that eluted from the HPLC column
were then analyzed for E2/D2-IsoPs by GC/MS and for
radioactivity (Fig.
6A). Radiolabeled PGE2 eluted in this system
between 16 and 18.5 min. Compounds representing endogenous
E2/D2-IsoPs were present that had the same retention
time upon GC as PGE2, but that eluted with different retention
volumes compared with PGE2 upon HPLC (1012, 1821, and
22.525 ml). Radiolabeled PGE2 eluted at a volume of
1.01.5 ml after unlabeled PGE2 using this HPLC solvent
system. Significantly, as shown in Fig.
6A, an endogenous E2/D2-IsoP peak
(indicated by the plus sign) was detected that coeluted with
unlabeled PGE2, suggesting that this compound is endogenously
derived rac-PGE2.
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The material that eluted from the HPLC column between 14.5 and 18.5 ml in Fig. 6A was subsequently subjected to reversed-phase HPLC using an isocratic solvent system of 30: 70:0.1 (v/v/v) acetonitrile/water/acetic acid. Aliquots of fractions collected were again analyzed for endogenous E2/D2-IsoPs by GC/MS and for radioactivity (Fig. 6B). Radiolabeled PGE2 eluted from the HPLC column with a retention volume of 28.532.5 ml. Analysis of aliquots of the eluted fractions by GC/MS showed that almost all of the unlabeled E2/D2-IsoP material detected in the chromatogram eluted at the retention volume of unlabeled PGE2 (29.533 ml), except for a small amount of additional material that eluted at 3739 ml.
Altering the polarity of a compound by derivatization and rechromatography of the compound can provide a powerful approach for purification and separation of biomolecules (9). Thus, the material that eluted from the HPLC column between 28.5 and 33 ml in Fig. 6B was converted to a PFB ester and rechromatographed on a normal-phase HPLC column using a solvent system of 92:8 (v/v) hexane/isopropyl alcohol. Fig. 6C shows the result of this HPLC step. Radiolabeled PGE2 eluted between 29.5 and 33.5 ml. A large peak representing endogenous E2/D2-IsoPs that coeluted with the PFB ester of unlabeled PGE2 was detected (27.531.5 ml).
Compounds that eluted from the HPLC column between 27.5 and 34 ml in Fig. 6C were then pooled. This material was subjected to further purification by reversed-phase HPLC using a solvent system of 51:49 (v/v) acetonitrile/water. The results of the analyses for radioactivity and endogenous E2/D2-IsoPs in the eluted fractions are shown in Fig. 6D. A single E2/D2-IsoP peak presumably representing rac-PGE2 was present that coeluted exactly with the PFB ester of unlabeled PGE2 (4244.5 ml). Radiolabeled PGE2 eluted slightly before the endogenous E2/D2-IsoP compound. Virtually identical results were obtained when putative rac-PGE2 generated from arachidonic acid oxidized in vitro was analyzed by the HPLC protocols described above.
Analysis of Endogenous Putative rac-PGE2
by GC/MSThe material that eluted between 40.5 and
44.5 ml upon the fourth HPLC step was then analyzed by GC/MS. As shown in
Fig. 7, two
E2/D2-IsoP peaks were present in the
m/z 524 chromatogram, representing the syn- and
anti-O-methyloxime isomers of putative
rac-PGE2. The amount of putative
rac-PGE2 present in this rat liver hydrolysate was 35
ng/1000 ng of total E2/D2-IsoP based upon losses of
[3H7]PGE2 that occurred with the four HPLC
purification steps. When the material indicated by peaks in the
m/z 524 chromatogram in
Fig. 7 was mixed with an
equivalent amount of derivatized synthetic PGE2, the two compounds
co-chromatographed perfectly upon capillary GC without any suggestion of a
shoulder on the GC peaks (data not shown). Additional experiments were
subsequently undertaken to confirm the identification of the compound in
Fig. 7 as PGE2.
First, analysis of the material as a deuterated O-methyloxime
derivative disclosed the presence of one carbonyl group. Second, analysis as a
deuterated trimethylsilyl ether derivative revealed that the compound had two
hydroxyl groups. Third, catalytic hydrogenation showed two double bonds
(16). Finally, treatment of
putative rac-PGE2 with 15% methanolic KOH for 30 min
converted it to a compound with a molecular weight and retention time
identical to those of PGB2 when analyzed by GC/MS
(Fig. 8)
(20). Taken together, these
findings strongly support the contention that the material represented in the
m/z 524 chromatogram in
Fig. 7 is
rac-PGE2.
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Analysis of Putative rac-PGE2 by Chiral HPLCAs noted, it is predicted that PGE2 generated by the IsoP pathway should be racemic. The HPLC steps utilized above to purify putative rac-PGE2 will not separate enantiomers. Thus, the compounds represented in the chromatogram in Fig. 7 were subjected to chiral column chromatography, and fractions that eluted from the HPLC column were analyzed by GC/MS. Fig. 9 shows the results of the analysis. The peak indicated by the asterisk co-chromatographed under these HPLC conditions with chemically synthesized PGE2, whereas the peak denoted by the plus sign co-chromatographed with chemically synthesized ent-PGE2. The material from each peak co-chromatographed perfectly with both PGE2 and ent-PGE2 upon GC and was indistinguishable upon MS analysis. Approximately equal amounts of the compounds were present, as would be expected. Furthermore, the ratio of methyloxime isomers of ent-PGE2 was essentially identical to that of PGE2. Taken together, these studies provide compelling evidence that PGE2 and ent-PGE2 are generated in vivo in significant quantities from the IsoP pathway. Essentially identical results were obtained from the analysis of putative rac-PGE2 formed from the peroxidation of arachidonate in vitro.
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Purification of Putative rac-PGD2 from Rat
Liver Hydrolysates by HPLCAs noted in
Fig. 5 (A and
B), chromatographic peaks were present that coeluted not
only with PGE2, but also with PGD2. We thus employed
similar approaches as those used to obtain evidence for the formation of
rac-PGE2 in vitro and in vivo to
determine whether rac-PGD2 is also generated.
Table I shows the HPLC
conditions utilized to purify putative rac-PGD2 and the
retention time of the compound at each step.
Fig. 10 illustrates the
results from GC/MS analysis of the material that eluted between 21 and 24 ml,
where PGD2 eluted, upon the fourth HPLC step. As shown, two
E2/D2-IsoP peaks were present in the
m/z 524 chromatogram, representing the syn- and
anti-O-methyloxime isomers of putative
rac-PGD2. The amount of putative
rac-PGD2 present in the rat liver hydrolysate from this
analysis was 55 ng/1000 ng of total E2/D2-IsoP,
based upon losses of [3H7]PGD2 that occurred
with the four HPLC purification steps. When the material denoted by the peaks
in the m/z 524 chromatogram in
Fig. 10 was mixed with an
equivalent amount of derivatized synthetic PGD2, the two compounds
co-chromatographed perfectly upon capillary GC without any suggestion of a
shoulder on the GC peaks (data not shown). Additional experiments confirmed
the identification of the compound in Fig.
10 as PGD2. First, analysis of the material as a
deuterated O-methyloxime derivative disclosed the presence of one
carbonyl group. Second, analysis as a deuterated trimethylsilyl ether
derivative revealed that the compound had two hydroxyl groups. Third,
catalytic hydrogenation showed two double bonds.
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Analysis of Putative rac-PGD2 by Chiral HPLCAs with PGE2 generated by the IsoP pathway, it is predicted that PGD2 should be racemic. Thus, the compounds represented in the m/z 524 chromatogram in Fig. 10 were subjected to chiral column chromatography, and fractions that eluted from the HPLC column were analyzed by GC/MS (Table I). Fig. 11 shows the results of the analysis. The peak indicated by the asterisk co-chromatographed under these HPLC conditions with chemically synthesized PGD2, whereas the peak denoted by the plus sign represents ent-PGD2. The material from each peak co-chromatographed perfectly with PGD2 upon GC and was indistinguishable upon MS analysis. Approximately equal amounts of compounds were present, as would be expected; and the relative amounts of methyloxime isomers of PGD2 and ent-PGD2 were very similar. Taken together, these studies provide strong evidence that PGD2 and ent-PGD2, in addition to PGE2 and ent-PGE2, are generated in vivo in significant quantities from the IsoP pathway. Again, essentially identical results were obtained from the analysis of putative rac-PGD2 formed from the peroxidation of arachidonate in vitro.
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Quantitative Analysis of rac-PGE2 and rac-PGD2 Generated in Vitro and in Rat LiverThe above studies provide substantial support for the hypothesis that PGE2 and PGD2 and their respective enantiomers can be generated via the IsoP pathway. We subsequently undertook experiments to determine the total amounts of rac-PGD2 and rac-PGE2 generated from the oxidation of arachidonate in vitro and in vivo in comparison with other E2/D2-IsoPs. These determinations are highly important because, based on previous reports (4), the vast majority (>90%) of endoperoxide intermediates generated by the autoxidation of polyunsaturated fatty acids have side chains that are cis in relation to the prostane ring. Indeed, we have recently confirmed that endoperoxides with cis-side chains predominate over trans-side chain compounds when arachidonate is oxidized in vitro (5). Thus, it would be predicted that the IsoP endoperoxide with a structure identical to PGH2 generated from the peroxidation of arachidonate in vitro and in vivo would compose a trivial fraction of the total endoperoxides that are formed. Therefore, the amounts of rac-PGE2 and rac-PGD2 that are subsequently generated from this endoperoxide intermediate would be present at no more than a few nanograms/1000 ng of total E2/D2-IsoPs (4). Employing the HPLC protocols utilized for the studies described above, we quantified rac-PGE2, rac-PGD2, and rac-15-E2t-IsoP in vitro and in vivo and also assessed the relative formation of each enantiomer in the racemic mixture. Losses of endogenous material during the chromatographic procedures were accounted for by determining the percent loss of the respective radiolabeled PG added to the samples prior to purification. As noted in Table II, the amounts of rac-PGE2 and rac-PGD2 far exceeded those predicted based upon the observations of O'Connor et al. (4), and the quantities of rac-PGE2 were at least as great as, if not greater than, those of rac-15-E2t-IsoP both in vitro and in vivo. In summary, these quantitative data provide support that PGE2 and PGD2 and their respective enantiomers are generated in significant amounts via the IsoP pathway.
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Excretion of Unesterified rac-PGE2 and
rac-PGD2 in Rat and Human Urine in
VivoIn addition to detecting the in vivo formation of
rac-PGE2 and rac-PGD2 esterified in
rat liver tissue, we also sought to determine whether these compounds are
present unesterified in human and rodent urine at base line and whether they
increase in association with oxidant stress.
Table III shows the results of
studies performed to determine the relative amounts of these eicosanoids under
these conditions. As shown, at baseline, the relative levels of both
ent-PGE2 and ent-PGD2 in humans and
rats were low and composed no more than 10% of the total
rac-PGE2 and rac-PGD2 generated. In
addition, in several of the urine samples obtained from normal humans and rats
at baseline, the levels of rac-PGE2 were below the limits
of assay detection. On the other hand, after treatment of rats with
CCl4, the levels of both ent-PGE2 and
ent-PGD2 increased significantly. This was particularly
the case for ent-PGD2. If one assumes that an amount of
PGD2 equivalent to that of ent-PGD2 is
generated via the IsoP pathway after administration of CCl4, then
15% of PGD2 present in rat urine under these conditions is
formed by a mechanism independent of COX. Analogously,
30% of
rac-PGD2 (total of PGD2 and
ent-PGD2) would thus be predicted to be generated by this
mechanism. Fig. 12 (A and
B) shows the results from chiral analysis of rat urine
for PGD2 and ent-PGD2 at base line and after
treatment with CCl4. As shown, at base line, the chromatographic
peak comprising PGD2 (*) in
Fig. 12A greatly
exceeded the enantiomer (+), suggesting that COX contributes to the vast
majority of PGD2 production at base line. After CCl4
administration (Fig.
12B), the levels of ent-PGD2 (+)
increased dramatically in relation to PGD2 (*), supporting the
contention that CCl4 has induced PG formation via the IsoP
pathway.
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To provide further evidence that PGs can be generated via the IsoP pathway,
we pretreated rats with indomethacin (10 mg/kg intraperitoneally for 24, 12,
and 1 h) prior to CCl4 administration and collected urine for 24 h
after the oxidant was given (6,
21).
Fig. 12C shows the
results from the chiral analysis of rac-PGD2. As shown,
the chromatographic peaks representing PGD2 and
ent-PGD2 are very similar. The levels of PGD2
and ent-PGD2 were 30% of those present in
CCl4-treated rats not given indomethacin
(Table III) and support the
contention that significant amounts of unesterified PGD2 (and to a
lesser extent, PGE2) can be formed by a mechanism independent of
COX in settings of oxidant stress.
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DISCUSSION |
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We propose that the formation of PGs independent of COX involves the
generation of two IsoP endoperoxide intermediates
(rac-15-H2t-IsoP and rac-15-H2c-IsoP)
that isomerize to rac-15-E2t-IsoP and
rac-15-D2c-IsoP, respectively. These eicosanoids
subsequently undergo rapid epimerization to compounds identical in all
respects to racemic PGE2 and PGD2, respectively
(Fig. 2). A number of lines of
evidence that we and others have obtained support this proposed mechanism of
formation. As noted, we have previously shown that IsoPs contain E/D-, F-, and
thromboxane-type prostane rings
(7). However, an important
distinction between IsoPs and PGs is that IsoP bicycloendoperoxide
intermediates contain side chains that are predominantly (>90%) oriented
cis in relation to the prostane ring
(4). Indeed, we have recently
confirmed that endoperoxides with cis-side chains predominate over
trans-side chain compounds when arachidonate is oxidized
(5). One IsoP that is formed in
abundance in vivo is 15-E2t-IsoP, which is generated from
the endoperoxide intermediate 15-H2t-IsoP
(10). It would also be
predicted that the endoperoxide 15-H2c-IsoP can rearrange to form
the analogous D-ring IsoP 15-D2c-IsoP. In contrast to other types
of prostanoids, E2/D2-IsoPs are
-hydroxyketone-containing compounds that can undergo reversible
keto-enol tautomerization under both acidic and basic conditions, allowing
rearrangement of the side chains that are initially cis to the more
stable trans-configuration. That the trans-configuration is
highly favored has been demonstrated by the finding that, when PGE2
is subjected to conditions that induce keto-enol tautomerism, <10% of the
compound rearranges to the cis-side chain isomer
15-E2t-IsoP (11).
Also, attempts to synthesize 15-D2c-IsoP have been unsuccessful
because epimerization at C-12 readily occurs during synthesis to yield
PGD2 (12).
In this study, we have shown that chemically synthesized 15-E2t-IsoP is unstable and rapidly epimerizes nonenzymatically to PGE2 in phosphate buffer at physiological pH. It is likely that the isomerization is further enhanced in the presence of protein-containing biological solutions, which have been shown to facilitate epimerization and dehydration of other eicosanoids (22). Whether the isomerization can be catalyzed enzymatically is unknown. That epimerization of IsoP endoperoxides occurred in the in vitro and in vivo studies reported herein is strongly supported by the fact that comparable amounts of rac-PGE2 and rac-15-E2t-IsoP were generated from the peroxidation of arachidonate. In addition, the abundance of rac-PGD2 lends credence to the hypothesis that epimerization occurs readily. As noted by our findings, the formation of rac-PGD2 predominates over that of rac-PGE2 both at base line and after oxidant stress, perhaps because this compound would be predicted to form more readily from the epimerization of rac-15-D2c-IsoP compared with PGE2 from 15-E2t-IsoP. In this study, the lack of a chemically synthesized 15-D2c-IsoP standard precludes our detection of this compound in vitro and in vivo, although it would be predicted that it would not be present in significant amounts.
Our results also suggest that epimerization of 15-E2t-IsoP and 15-D2c-IsoP to PGE2 and PGD2, respectively, occurs to a significant extent while these compounds are esterified in phospholipids based on two lines of evidence. First, a pattern of peaks virtually identical to that shown in the chromatograms in Fig. 5 was obtained after hydrolysis of rat liver phospholipids that had been treated with methyloxime HCl prior to hydrolysis. Second, in control experiments, the conversion of exogenously added 15-E2t-IsoP to PGE2 occurred to a negligible extent during sample workup.
A number of important physiological and pharmacological issues emerge from the this study. The first relates to the fact that formation of bioactive PGs occurs in vivo to a significant extent via the IsoP pathway in settings of oxidative stress and potentially in other inflammatory situations. Although levels of PGs derived via this mechanism are low at base line in normal humans and animals, they represent up to 15% of PGD2 present in the urine of rats treated with CCl4, and these PGs are formed independent of COX inhibition. IsoPs have been implicated as mediators of oxidant stress (2325). Thus, it will be important to investigate the extent to which not only IsoPs, but PGs, contribute to adverse sequelae of oxidative injury.
Although the biological properties of PGE2 and PGD2 have been well characterized (1), our studies suggest that equal amounts of the enantiomers of these PGs are also produced. It will thus be of interest to explore the bioactivity of ent-PGE2 and ent-PGD2. In this respect, the former compound was synthesized for the studies reported herein, and experiments to determine its biological relevance will likely yield important insights into its role in oxidative injury.
The metabolism of PGE2 and PGD2 has been extensively
studied in animals and humans
(1). The metabolism of parent
PGs via the formation of C-13,14-dihydro-15-keto derivatives and subsequent
- or
-oxidation generally renders them inactive. However, this is
not the case for the one IsoP whose metabolism has been studied in detail,
15-F2t-IsoP. The major metabolite of this compound is
2,3-dinor-5,6-dihydro-15-F2t-IsoP, which results from one step of
-oxidation and an unusual C-5C-6 double bond reduction
(26). Interestingly, this
metabolite displays bioactivity as a vasoconstrictor similar to that of
15-F2t-IsoP (27).
Thus, studying the metabolism of ent-PGs, in addition to their
biological activities, may provide important insights into their role as
mediators of oxidant stress. In this regard, we have recently found that,
unlike PGE2, ent-PGE2 is a poor substrate for
15-hydroxyprostaglandin dehydrogenase, suggesting that the metabolism of this
eicosanoid is significantly different from that
PGE2.2
The studies reported herein are highly relevant with regard to human pharmacology in that they suggest that a second pathway operates in vivo to generate PGs and is independent of COX. That this pathway contributes to the formation of PGs in settings of oxidant stress has been discussed above. On the other hand, the extent to which it contributes to PG production in other disease states or at base line has not been elucidated. Administration of nonsteroidal anti-inflammatory agents to humans has been shown to significantly decrease production of PGs and PG metabolites, although the degree of suppression varies depending on the eicosanoid measured. For example, administration of high doses of nonsteroids (e.g. 1.5 g of aspirin or more or the equivalent) to normal human volunteers is associated with a 90% reduction in thromboxane formation and a >80% reduction in PGI2 (2830). In contrast, the same doses of these agents have been reported to be associated with no greater than a 60% decrease in PGE2 excretion (28). In this regard, we have made similar observations (31). The reasons for this discrepancy are unknown; but in light of our findings that PGs, particularly PGE2, are formed via a non-COX mechanism, it is intriguing to postulate that part of the reason that aspirin-like drugs fail to inhibit PGE2 production compared with other PGs in certain settings is that the former compound can be produced from IsoP intermediates.
In summary, we report that a second pathway exists for the formation of bioactive PGs in vivo that is independent of COX. This finding is likely of physiological and pharmacological importance because it would be predicted that the generation of PGs via this mechanism would not be inhibited by aspirin or other COX inhibitors. The extent to which formation of PGs independent of COX contributes to human physiology and pathophysiology remains to be elucidated.
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FOOTNOTES |
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Recipient of a Burroughs Wellcome Fund Clinical Scientist Award in
Translational Research. To whom correspondence should be addressed: Vanderbilt
University, 526 RRB, 23rd and Pierce Aves., Nashville, TN 37232-6602. Tel.:
615-343-1124; Fax: 615-322-3669; E-mail:
jason.morrow{at}mcmail.vanderbilt.edu.
1 The abbreviations used are: COX, cyclooxygenase; PG, prostaglandin; IsoP,
isoprostane; GC, gas chromatography; MS, mass spectrometry; ent-,
enantiomeric; rac-, racemic; PFB, pentafluorobenzyl; HPLC, high
pressure liquid chromatography.
2 L. Gao, W. E. Zackert, J. J. Hasford, M. E. Danekis, G. L. Milne, C.
Remmert, J. Reese, H. Yin, H.-H. Tai, S. K. Dey, N. A. Porter, and J. D.
Morrow, unpublished data.
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ACKNOWLEDGMENTS |
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REFERENCES |
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