Formation of Prostaglandins E2 and D2 via the Isoprostane Pathway

A MECHANISM FOR THE GENERATION OF BIOACTIVE PROSTAGLANDINS INDEPENDENT OF CYCLOOXYGENASE*

Ling Gao {ddagger} §, William E. Zackert §, Justin J. Hasford §, Michael E. Danekis §, Ginger L. Milne §, Catha Remmert §, Jeff Reese ¶, Huiyong Yin ||, Hsin-Hsiung Tai **, Sudhansu K. Dey ¶, Ned A. Porter || and Jason D. Morrow {ddagger} § {ddagger}{ddagger}

From the Departments of {ddagger}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.


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
It has heretofore been assumed that the cyclooxygenases (COXs) are solely responsible for peostaglandin (PG) synthesis in vivo. An important structural feature of PGH2 formed by COX is the trans-configuration of side chains relative to the prostane ring. Previously, we reported that a series of PG-like compounds termed isoprostanes (IsoPs) are formed in vivo in humans from the free radical-catalyzed peroxidation of arachidonate independent of COX. A major difference between these compounds and PGs is that IsoPs are formed from endoperoxide intermediates, the vast majority of which contain side chains that are cis relative to the prostane ring. In addition, unlike the formation of eicosanoids from COX, IsoPs are formed as racemic mixtures because they are generated nonenzymatically. IsoPs containing E- and D-type prostane rings (E2/D2-IsoPs) are one class of IsoPs formed, and we have reported previously that one of the major IsoPs generated is 15-E2t-IsoP (8-iso-PGE2). Unlike PGE2, 15-E2t-IsoP is significantly more unstable in buffered solutions in vitro and undergoes epimerization to PGE2. Analogously, the D-ring IsoP (15-D2c-IsoP) would be predicted to rearrange to PGD2. We now report that compounds identical in all respects to PGE2 and PGD2 and their respective enantiomers are generated in vivo via the IsoP pathway, presumably by epimerization of racemic 15-E2t-IsoP and 15-D2c-IsoP, respectively. Racemic PGE2 and PGD2 were present esterified in phospholipids derived from liver tissue from rats exposed to oxidant stress at levels of 24 ± 16 and 37 ± 12 ng/g of tissue, respectively. In addition, racemic PGs, particularly PGD2, were present unesterified in urine from normal animals and humans and represented up to 10% of the total PG detected. Levels of racemic PGD2 increased 35-fold after treatment of rats with carbon tetrachloride to induce oxidant stress. In this setting, PGD2 and its enantiomer generated by the IsoP pathway represented ~30% of the total PGD2 present in urine. These findings strongly support the contention that a second pathway exists for the formation of bioactive PGs in vivo that is independent of COX.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Cyclooxygenase (COX)1-1 and COX-2 catalyze the committed steps in formation of prostaglandins (PGs) by generating the unstable bicycloendoperoxide intermediate PGH2 (13). PGH2 is subsequently metabolized to the parent eicosanoids PGE2, PGD2, PGF2{alpha}, PGI2, and thromboxane A2, which exert a plethora of biological activities (1). The formation of PGH2 is stereospecific in that, among other structural aspects, the side chains of PGH2 are oriented in the trans-configuration relative to the prostane ring (Fig. 1A). This conformation is highly favored thermodynamically (4, 5).



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FIG. 1.
Structure of the bicycloendoperoxide intermediate PGH2 (A) derived from COX and structures of the IsoP endoperoxides 15-H2t-IsoP (B) and 15-H2c-IsoP (C).

 

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{alpha}) 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 {beta}-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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FIG. 2.
Base-catalyzed mechanism of epimerization of 15-E2t-IsoP to PGE2 (A) and 15-D2c-IsoP to PGD2 (B) involving keto-enol tautomerization. Acid-catalyzed epimerization can also occur. R = either hydrogen or phospholipids depending on whether the IsoPs epimerize as free acids or esterified in phospholipids.

 

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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FIG. 3.
Structures of PGE2 and PGD2 and their respective enantiomers.

 


    EXPERIMENTAL PROCEDURES
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Materials—Arachidonic acid, dimethylformamide, CCl4, and undecane were purchased from Aldrich. Pentafluorobenzyl (PFB) bromide, methoxyamine HCl, diisopropylethylamine, and Apis mellifera venom phospholipase A2 were from Sigma. [2H3]Methoxyamine HCl was from Cambridge Isotope Laboratories, Inc. (Andover, MA). N,O-Bis(trimethylsilyl)trifluoroacetamide was from Supelco Inc. (Bellefonte, PA). N,O-[2H9]Bis(trimethylsilyl)acetamide was from CDN Isotopes (Pointe-Claire, Quebec, Canada). Organic solvents were from EM Science (Darmstadt, Germany). C18 and silica Sep-Pak cartridges were from Waters Associates (Milford, MA). 60ALK6D TLC plates were from Whatman (Maidstone, UK). [2H4]PGE2, [2H4]PGD2, and [2H4]PGB2 were from Cayman Chemical Co., Inc. (Ann Arbor, MI). [3H7]PGE2 and [3H7]PGD2 (~180–200 Ci/mmol) were obtained from Amersham Biosciences. 15-E2t-[3H6]IsoP (160 Ci/mmol) was commercially prepared from [3H7]PGE2 by Amersham Biosciences as described (10). The enantiomer of PGE2 (ent-PGE2) was synthesized by Dr. Doug Taber (University of Delaware) (15).

Epimerization of 15-E2t-IsoP in Phosphate Buffer—15-E2t-IsoP (1 µM) was incubated in 50 mM KPO4 buffer, pH 7.4, for 0–24 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 Acid—Arachidonic 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 Urine—A 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, 1–4 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 IsoPs—Depending 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 ~0.5–3 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 (~15–40 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 (1–2.5 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/MS—Quantification 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).


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Epimerization of 15-E2t-IsoP in Phosphate Buffer—We initially determined the extent to which 15-E2t-IsoP undergoes epimerization to PGE2 in a buffered solution at physiological pH (50 mM KPO4, pH 7.4). Products that were quantified included the starting material 15-E2t-IsoP and PGE2 and their respective dehydration products, 15-A2t-IsoP and PGA2. Amounts are expressed as percent of total PG at a particular time point. The identification of PGE2 was also confirmed by NMR comparison with a chemically pure PGE2 standard. The results are shown in Fig. 4. As shown, 15-E2t-IsoP epimerized in a time-dependent manner to PGE2. The half-life for this conversion under the conditions noted was ~2 h. In addition, small amounts of 15-A2t-IsoP and PGA2 were formed, presumably as a result of dehydration of 15-E2t-IsoP and PGE2, respectively. These findings support the hypothesis that E2/D2-IsoPs can readily rearrange to E/D-ring PGs in aqueous environments.



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FIG. 4.
Time course of epimerization of 15-E2t-IsoP in KPO4 buffer, pH 7.4. Data are expressed as means ± S.D.

 

Analysis of E2/D2-IsoPs from the Oxidation of Arachidonic Acid in Vitro and in Vivo—Fig. 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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FIG. 5.
A, analysis of arachidonic acid oxidized in vitro for E2/D2-IsoPs by GC/negative ion chemical ionization MS; B, analysis of hydrolyzed lipid extracts from livers of rats treated with CCl4 for E2/D2-IsoPs by GC/negative ion chemical ionization MS. The two peaks in the m/z 528 ion current chromatograms represent syn- and anti-O-methyloxime isomers of the [2H4]PGE2 internal standard. A series of peaks representing E2/D2-IsoPs are in the m/z 524 chromatograms. Compounds denoted by asterisks co-chromatographed with the O-methyloxime isomers of PGE2, whereas those denoted by the plus signs co-chromatographed with the O-methyloxime isomers of PGD2. Analyses were performed on separate days, accounting for differences in GC retention time.

 

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 ~15–20% 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 ~5–10 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 HPLC—We 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 (10–12, 18–21, and 22.5–25 ml). Radiolabeled PGE2 eluted at a volume of ~1.0–1.5 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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FIG. 6.
HPLC analysis of a mixture of E2/D2-IsoPs from hydrolyzed rat liver phospholipids for rac-PGE2 in Fig. 5B. Details regarding solvent systems used are described under "Experimental Procedures." Tritiated PGE2 was added to the mixture at the beginning, and aliquots of eluted fractions were assayed for radioactivity (•). Aliquots were also assayed and quantified by GC/MS for the presence of E2/D2-IsoP peaks with the same retention time as authentic PGE2 ({blacktriangleup}). All HPLC purifications were carried out isocratically. A, normal-phase HPLC as free acids of the initial mixture of E2/D2-IsoPs as shown in Fig. 5B. The plus sign denotes fractions in which chemically pure unlabeled PGE2 eluted using this solvent system. B, reversed-phase HPLC as free acids of the material that eluted at the retention volume between 14.5 and 18.5 ml in A. C, normal-phase HPLC as PFB esters of the material that eluted at the retention volume between 28.5 and 33 ml in B. D, reversed-phase HPLC as PFB esters of the material that eluted at the retention volume between 27.5 and 34 ml in C.

 

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 28.5–32.5 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 (29.5–33 ml), except for a small amount of additional material that eluted at 37–39 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 (27.5–31.5 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 (42–44.5 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/MS—The 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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FIG. 7.
Selected ion current chromatogram obtained from the GC/MS analysis of the material that eluted at a retention volume between 40.5 and 44.5 ml in Fig. 6D. Only a single set of m/z 524 peaks representing the syn- and anti-O-methyloxime isomers of endogenous putative rac-PGE2 remained after the four HPLC purification procedures shown in Fig. 6. The peaks in the m/z 528 chromatogram represent the syn- and anti-O-methyloxime isomers of the deuterated PGE2 internal standard. The amount of putative rac-PGE2 in the fraction analyzed was ~35 ng/1000 ng of total E2/D2-IsoP.

 


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FIG. 8.
Selected ion current chromatogram from the GC/MS analysis of putative rac-PGE2 in Fig. 7 after treatment with methanolic KOH. In the lower m/z 438 chromatogram is a single peak representing the [2H4]PGB2 internal standard. In the upper m/z 434 chromatogram is a single peak that co-chromatographed perfectly with unlabeled PGB2 and that represents endogenous rac-PGB2.

 

Analysis of Putative rac-PGE2 by Chiral HPLC—As 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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FIG. 9.
Chiral HPLC separation of rac-PGE2. The putative rac-PGE2 purified as described in the legend to Fig. 6 was subjected to chiral column chromatography using the solvent system 93:7 (v/v) hexane/isopropyl alcohol. Aliquots of fractions that eluted from the HPLC column were then analyzed for PGE2 by GC/MS. The peak indicated by the asterisk co-chromatographed in the HPLC solvent system with chemically pure PGE2, whereas the peak denoted by the plus sign coeluted with ent-PGE2.

 

Purification of Putative rac-PGD2 from Rat Liver Hydrolysates by HPLC—As 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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TABLE I
HPLC retention times for purification of putatitive rac-PGD2 and chiral separation of enantiomers

See "Experimental Procedures" for details regarding the columns used and solvent conditions employed for each HPLC step.

 


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FIG. 10.
Selected ion current chromatogram obtained from the GC/MS analysis of the material that eluted at a retention volume of 21–24 ml after the fourth HPLC step to purify rac-PGD2 as noted in Table I. Only a single set of m/z 524 peaks representing the syn- and anti-O-methyloxime isomers of endogenous putative rac-PGD2 remained after the four HPLC purification procedures shown in Table I. The peaks in the m/z 528 chromatogram represent the syn- and anti-O-methyloxime isomers of the deuterated PGD2 internal standard. The amount of putative rac-PGE2 in the fraction analyzed was ~55 ng/1000 ng of total E2/D2-IsoP.

 

Analysis of Putative rac-PGD2 by Chiral HPLC—As 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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FIG. 11.
Chiral HPLC separation of rac-PGD2. The putative rac-PGD2 purified as noted in Table I was subjected to chiral column chromatography using the solvent system 95:5 (v/v) hexane/isopropyl alcohol. Aliquots of fractions that eluted from the HPLC column were then analyzed for PGD2 by GC/MS. The peak indicated by the asterisk co-chromatographed in the HPLC solvent system with chemically pure PGD2, whereas the peak denoted by the plus sign represents ent-PGD2.

 

Quantitative Analysis of rac-PGE2 and rac-PGD2 Generated in Vitro and in Rat Liver—The 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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TABLE II
Levels of rac-PGE2, rac-PGD2, and rac-15-F2t-IsoP generated from the peroxidation of arachidonic acid in vitro and in liver hydrolysates from rats after treatment with CCl4

Data are expressed as means ± S.D.

 

Excretion of Unesterified rac-PGE2 and rac-PGD2 in Rat and Human Urine in Vivo—In 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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TABLE III
Levels of PGE2, ent-PGE2, PGD2, and ent-PGD2 in urine from rats and humans

Data are expressed as means ± S.D. (n = 8).

 


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FIG. 12.
Chiral analysis of rat urine for rac-PGD2. The putative rac-PGD2 in each case was purified as noted in Table I and under "Results" and subjected to chiral column chromatography using the solvent system 95:5 (v/v) hexane/isopropyl alcohol. Aliquots of fractions that eluted from the HPLC column were then analyzed for PGD2 by GC/MS. The peaks indicated by the asterisks co-chromatographed in the HPLC solvent system with chemically pure PGD2, whereas the peaks denoted by the plus signs represent ent-PGD2. A, chiral analysis of rac-PGD2 in urine from a normal rat. The predominant stereoisomer is PGD2. B, chiral analysis of rac-PGD2 in urine from a rat treated with CCl4 (2 ml/kg) to induce oxidant stress. As shown, the relative height of the chromatographic peak representing ent-PGD2 compared with PGD2 is significantly higher than that in A. C, chiral analysis of rac-PGD2 in urine from a rat pretreated with indomethacin prior to receiving CCl4. Although the relative height of the peak representing PGD2 decreased significantly compared with that in B, the height of the peak representing ent-PGD2 was largely unchanged. See Table III for details.

 

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.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
This study describes the formation of PGE2 and PGD2 independent of COX and involving the free radical-catalyzed peroxidation of arachidonate. We have reported that significant amounts of rac-PGE2 and rac-PGD2 are generated in vitro and in vivo in settings of oxidant stress. Unlike PGs formed via COX, generation of eicosanoids by this mechanism results in the formation of compounds as racemic mixtures because the oxygenation of arachidonic acid does not occur stereospecifically (2, 3, 6). Our initial interest in determining whether PGs are generated via the IsoP pathway emerged from the observation that compounds with the same molecular weights and GC retention times as PGE2 and PGD2 are present when analyzed by GC/MS in mixtures of arachidonate oxidized in vitro and in rat liver hydrolysates. Utilizing a variety of high resolving chromatographic, chemical, and mass spectrometric approaches, we have found that substantial quantities of these racemic PGs can be generated. Analysis of putative rac-PGE2 and rac-PGD2 by chiral HPLC revealed that each compound is composed of two enantiomers generated in equal amounts in vitro and in liver tissue from rats exposed to oxidant stress. rac-PGE2 and rac-PGD2 were also present in the unesterified form in significant amounts in urine from rats treated with CCl4, and their formation was unaffected by COX inhibition. That COX is not involved in the formation of these compounds is also supported by the findings that these PGs could be generated in vitro without COX and were present in vivo esterified in phospholipids. COX is not active on arachidonate esterified in phospholipids (1). Finally, compounds with retention times and molecular weights identical to those of PGE2 and PGD2 were present when liver tissue from COX-1//COX-2/ mice was analyzed for E2/D2-IsoPs.

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 {beta}-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 {beta}- or {omega}-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 {beta}-oxidation and an unusual C-5–C-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.


    FOOTNOTES
 
* This work was supported in part by National Institutes of Health Grants DK48831, GM42056, CA77839, HD12304, and HL46296. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{ddagger}{ddagger} 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. Back

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. Back


    ACKNOWLEDGMENTS
 
We thank Drs. Jack Roberts and Doug Taber for helpful discussions.



    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

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