©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Cylooxygenase-dependent Formation of the Isoprostane, 8-Epi Prostaglandin F(*)

Domenico Pratico , John A. Lawson , Garret A. FitzGerald (§)

From the (1) Center for Experimental Therapeutics, The University of Pennsylvania, Philadelphia, Pennsylvania 19104

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

Isoprostanes are a family of prostaglandin (PG) isomers formed in an enzyme-independent manner. They circulate in plasma and are excreted in urine. One of them, 8-epi PGFis a vasoconstrictor and mitogen, effects which are prevented by thromboxane antagonists. Given that 8-epi PGFmay be formed by cyclooxygenase (COX) (Corey, E. J., Shih, C., Shig, N-Y., and Shimoji, K. (1984) Tetrahedron Letts. 44, 5013-5016; Hecker, M., Ullrich, V., Fischer, C., and Meese, C. O. (1987) Eur J. Biochem. 169, 113-123) and that this might confound its use as an index of free radical generation, we sought to characterize the mechanism of its formation by human platelets.

Activation of platelets by threshold concentrations of collagen, thrombin, and arachidonic acid resulted in formation of 8-epi PGFcoincident with that of the COX product, thromboxane, and the 12 lipoxygenase product, 12-hydroxyeicosatetraenoic acid, as detected by selected ion monitoring assays using gas chromatography-mass spectrometry. The effect appeared selective for 8-epi PGFamong the Fisoprostanes. Pretreatment of platelets with aspirin or indomethacin abolished 8-epi PGFformation. COX-independent activation of platelets by high doses of collagen or thrombin, by the phorbol ester, phorbol 12-myristate 13-acetate, or the prostaglandin endoperoxide analog, U 46619 was not associated with 8-epi PGFformation.

Confirmation of the nature of the material formed by platelet COX as 8-epi PGFincluded its cochromatography over three highly resolving high performance liquid chromatography systems, identification by electron impact mass spectrometry, and its formation by partially purified COX. Inhibition of platelet thromboxane formation was associated with augmented 8-epi PGFformation.A major component of 8-epi PGFformed in serum by healthy volunteers was shown to be sensitive to inhibition by aspirin ex vivo.

In addition to its generation by free radical catalyzed mechanisms, 8-epi PGFmay be formed as a PG by human platelets. Given that activation of platelet COX characterizes many of the human syndromes which are putatively associated with free radical generation, assessment of the contribution of this pathway is relevant to the use of 8-epi PGFas an index of lipid peroxidation in vivo.


INTRODUCTION

The isoprostanes are families of prostaglandin isomers formed in a free radical catalyzed manner from arachidonic acid (1, 2) . They are produced via peroxyl radical isomers which undergo endocylcization and subsequent reduction; isoprostanes of the E and F series have been reported. Given the manner of their formation, it has been proposed that measurement of isoprostanes might offer a quantitative index of free radical generation in vivo. Existing indices of this process are of controversial validity when applied to clinical studies. Consequently, our understanding of the role of free radicals in human disease has been confused (3, 4) , and there is a paucity of data underlying the selection and usage of antioxidant drugs in vivo. Particular attention has focused on one of the isoprostanes, 8-epi PGF.() This compound is among the most abundant of the Fisoprostanes formed under physiological conditions in humans (5) and induces vasoconstriction in the renal and pulmonary circulations (6, 7) , mitogenesis in NIH 3T3 cells (8) , and platelet shape change, but not aggregation and the release reaction (9, 10) . These effects are prevented by thromboxane receptor antagonists. We have developed a sensitive and specific assay for this compound and have shown its excretion in human urine to be increased during coronary reperfusion and in chronic cigarette smokers, both settings putatively associated with increased free radical generation (11, 12) . However, activation of platelet cyclooxygenase (COX), as reflected by increased excretion of thromboxane metabolites, also characterizes these conditions (13, 14) . COX-dependent formation of 8-epi PGFmight confound its usefulness as an analytical target reflective of free radical formation in vivo, particularly in the setting of coincident platelet activation. Thus, we addressed the hypothesis that this compound may be a product of COX metabolism of endogenous arachidonic acid in human platelets.


MATERIALS AND METHODS

Human Subjects The study was scrutinized and approved by the Ethics Committee of the Mater Hospital, Dublin, Ireland. All participants signed an informed consent document prior to participation in the study. The volunteers were Caucasian males, aged 21-49 years. All were nonsmokers and abstained from all medication for at least 14 days prior to participation in the study. No abnormality was revealed on clinical examination or on full blood count, platelet count, or routine biochemical screening. Blood was collected, without stasis, into a plastic syringe, using a 21-gauge needle from an antecubital vein after the subject had been sitting for at least 5 min. Samples were drawn into 3.8% sodium citrate in a ratio of 9:1. The samples were taken between 9:00 a.m. and 11:00 a.m. at least 12 h after the last meal.

Studies of Platelet Function

Platelets were harvested as described previously (15). Briefly, platelet-rich plasma (PRP) was prepared by centrifugation of the blood sample at 160 g for 10 min and platelet-poor plasma (PPP) by centrifugation of PRP at 900 g for 10 min at room temperature. Washed platelets (WP) were isolated from PRP after centrifugation and resuspended in calcium and magnesium-free Hank's balanced salt solution at pH 7.4, containing 10% autologous PPP. Platelet number was adjusted to 3 10platelets/ml with PPP or Hank's balanced salt solution. Platelet aggregation was studied either in PRP or in WP by using a PAP-4 model BIO-DATA aggregometer (BIO-DATA Corporation, Hatboro, PA), at 37 °C, in siliconized cuvettes with continuous stirring. Platelet aggregation was performed using threshold concentration (TC) of agonists, defined as the lowest concentration that gave an irreversible aggregation tracing with an amplitude between 65 and 85% of maximal.

The role of COX was assessed by preincubating PRP or WP for 5 min at 37 °C with aspirin (100 µM; Aspidol, Maggioni, Milan, Italy) or indomethacin (10 µM; Sigma) and inhibition of activity confirmed by the absence of an aggregation response to arachidonic acid (1 mM; Cayman Chemical Co., Ann Arbor, MI). The mechanism of 8-epi PGFformation was also explored under similar conditions using vitamin E (500 µM),mannitol (5 mM), deoxyribose (5 mM; all from Sigma). We also used the thromboxane synthase inhibitors (16, 17) ,1(7-carboxyheptyl)imidazole-HCI (10 µM) and sodium furegrelate (U-63557A, 50 µM) (Cascade Biochem., Berkshire, United Kingdom). Solid-phase Extraction (SPE), Thin Layer Chromatography (TLC), and Derivatization Briefly, product formation was stopped at fixed times after addition of the platelet agonists by adding glacial acetic acid and lowering the pH to 3-3.5. Tetra and hepta deuterated internal standards of TxBand 12-HETE (Cayman Chemical Co., Ann Arbor, MI), respectively, were then added to the platelet preparation and the samples applied to a 100-mg octadecylsilyl (ODS) solid-phase extraction column (Alltech Associates Inc., Deerfield MI) that had been prepared as per the manufacturer's instructions. The column was then washed with 25% methanol, 75% water and 100% hexane, dried, then eluted with 100% ethyl acetate. The samples were initially derivatized as pentafluorobenzyl (PFB) esters by adding 10 µl of diisopropylethylamine and 20 µl of 10% PFB Br in acetonitrile and allowing the reaction to proceed for at least 10 min at room temperature. This reaction mixture was dried under nitrogen and applied to a TLC plate (LK6D, 60A Silica Gel Plates Whatman Inc., Clifton NJ). The mobile phase was 100% ethyl acetate. The extractions were then dried under a stream of nitrogen, the trimethylsilyl ether derivative was formed by adding 10 µl of BSTFA Supelco Inc., Bellafonte, PA) and 10 µl of pyridine and allowing the reaction to proceed for 10 min at room temperature. The reaction mixture was then dried under nitrogen, the sample redissolved in 20 µl of dodecane, and analyzed by GC-MS. 8-Epi PGFwas measured using an O-labeled internal standard derivatized as the PFB ester as described above. The reaction mixture was dried under nitrogen and applied to a TLC plate (LK6D, 60A Silica Gel Plates). The mobile phase was 80% ethyl acetate and 20% heptane. The extractions were dried under a stream of nitrogen and the tert-butyldimethylsilyl ether derivative formed by adding 10 µl of MTBSTFA (Sigma) and 10 µl of pyridine, and allowing the reaction to proceed for 24 h at room temperature. The reaction mixture was then dried under nitrogen, the sample redissolved in 20 µl of dodecane, and analyzed by GC-MS.

For electron impact-mass spectrometry (EI/MS) studies, the methyl ester was formed by dissolving the sample in 100 µl of methanol and 500 µl of a dilute solution of diazomethane in ether and allowing it to stand at room temperature for 30 min. GC-MS All GC-MS studies were performed on a Delsi-Nermag Automass 150 (Delsi-Nermag, Argenteuil, France) equipped with a Varian 1077 split/splitless injector operated in the splitless mode, at 280 °C. Helium was used as the carrier gas. The interface was maintained at 280 °C, the ion source at 250 °C. The MS was operated in the negative ion, chemical ionization mode, utilizing methane as the reagent gas. The ion source was maintained at 250 °C. For electron impact studies, the ionization energy was 70 eV, and the source temperature was 190 °C.

8-Epi PGF

A 30-µm DB-1 capillary column of 0.25 mm inner diameter with 0.25 µm of coating was utilized. The temperature program was 190-320° at 20 °C/min. The retention time was approximately 17 min. A 40-m DB-1 0.18 mm inner diameter, 0.4-µm coating was used for studies aimed at examining the purity of the 8-epi PGF, using the same temperature program. The retention time on this system was approximately 1 h. Integration times for selected ion monitoring studies were 500 ms for each of the two ions monitored; m/z 695 for 8-epi PGFand m/z 699 for [O]8-epi PGF.

TxB

Platelet TXAwas measured as its hydrolysis product, TxB. A 15-m DB-1 capillary column of 0.25 mm inner diameter with 0.25 µm of coating was used; the temperature program was as above described for 8-epi PGF. The retention time was approximately 7 min, and ions monitored were m/z 614 for TxBand m/z 618 for [H]TxB.

12-HETE

A 10-m DB-1 column of 0.25 mm inner diameter with 0.25 µm of coating was used with a temperature program as described above. The retention time was approximately 6 min, and the ions monitored were m/z 391 for 12-HETE and m/z 399 for [H]12-HETE. High Performance Liquid Chromatography (HPLC) A Hewlett-Packard (HP)1050 HPLC in line with an HP 1050 UV detector and a Flo-One Radioactivity detector (Radiomatic Instruments, Meriden, CT) was used for all HPLC experiments. Straight phase (SP) chromatography was performed on an Ultrasphere Si 5-µm column, 4.6 mm inner diameter 25 cm (Beckman Instruments, Fullerton, CA). Reverse phase (RP) chromatography utilized an Ultrasphere ODS 5-µm column, 4.6 mm 25 cm. The flow rate was 1 ml/min in all experiments. Incubation of Arachidonic Acid (AA) with Partially Purified COX-1 AA (10 µg) or [H]AA (10 µCi) in 20 µl of methanol was added to 1 ml of Tris buffer, pH 8, containing 2 nM phenol, 1 mM EDTA, and 500 units of COX-1. The reaction was allowed to proceed for 1 min at 37 °C, at which time 150 µl of glacial acetic acid and 3 ml of ether containing 1 mg of triphenyl phosphine was added. After vortexing and centrifugation, the ether was removed, dried under N, and applied to a TLC plate which was then developed with a mobile phase of 10% methanol, 90% ethyl acetate, 0.1% glacial acetic acid. A 1-cm zone centered on 8-epi PGFwas scraped, extracted, and further purified by HPLC on an ODS column utilizing a mobile phase of 25% acetonitrile, 75% water, 0.1% glacial acetic acid. The elution volume was 24-26 ml. Ex Vivo Study Four healthy volunteers (age 30 ± 8 years), who had not taken any medication during the previous 2 weeks, were given 1 g of aspirin. All were nonsmokers. Blood samples were taken before aspirin (base line), at 3 h and at 24 h after the ingestion and the serum was analyzed for TxBand 8-epi PGF. Statistical Analysis Data were initially analyzed using analysis of variance. Pairwise comparisons were made using the Student's t test, where appropriate. Data are displayed as mean ± standard deviation.


RESULTS

Formation of Arachidonic Acid Products by Human Platelets

All three products, TxB, 12-HETE, and 8-epi PGFwere below the detection levels of their corresponding assays (2 ng/ml, 2 ng/ml, and 2 pg/ml, respectively) in unstimulated PRP and WP.

Threshold concentrations of collagen (0.5-2 µg/ml) induced irreversible aggregation in PRP after a lag phase of 30 s. Three min after addition of agonist, the levels of TxB(130 ± 20 ng/ml) and 8-epi PGF(80 ± 15 pg/ml) had risen dramatically ( n = 5; p < 0.00 1). Addition of aspirin or indomethacin 5 min prior to agonist completely prevented formation of both TxBand 8-epi PGF. Consistant with this observation, threshold concentrations of peroxide-free arachidonic acid (20-50 µM) also increased TxB(400 ± 25 ng/ml) and 8-epi PGF(350 ± 30 pg/ml) which were both inhibited by the COX inhibitors ( n = 4).

We studied WP to extend these observations and incorporated measurement of 12-HETE as a non-COX, enzymatic product of arachidonic acid in platelets. Collagen increased all three products in WP coincident with aggregation. Aspirin pretreatment abolished formation of TxBand 8-epi PGF; it also caused 12-HETE formation to fall from 320 to 140 ng/ml ( p < 0.001). Formation of the three products appeared related in time; an initial lag phase in the first minute preceded initiation of aggregation, which was maximal 3 min after addition of agonist when product formation had reached a plateau (Fig. 1). Interestingly, the conditions of the assay allowed chromatographic separation of an endogenous peak corresponding to the retention time of authentic O-labeled 8-epi PGFfrom peaks which probably correspond to other Fisoprostanes (Fig. 2, center panel). Although internal standards for the other compounds were not included in the assay, the agonist-induced increments appeared selective for the peak comigrating with the 8-epi PGFinternal standard irrespective of the platelet agonist (Fig. 2, lower panel). A similar, apparently selective increase in 8-epi PGFformation was observed when aggregation was induced by threshold concentration of thrombin (0.1-0.3 unit/ml). The pattern was similar to that observed with collagen, although the rise in 8-epi PGFappeared to precede that of the other two products (Fig. 3 A). Again, aspirin completely suppressed TxBand 8-epi PGFand partially suppressed 12-HETE formation. Similar results were obtained with threshold concentrations (1-3 µM) of the calcium ionophore A23187 (Fig. 3 B).


Figure 1: Time course of eicosanoid formation in collagen-stimulated washed human platelets. Significant ( p < 0.001) increases occurred coincident with aggregation in the predominant COX product TxB(), the predominant 12-lipoxygenase product, 12-HETE (), and 8-epi PGF(). Pretreatment of platelets with aspirin (asa 100 µM) prevented aggregation and completely inhibited TxBand 8-epi PGFformation. Production of 12-HETE was significantly ( p < 0.00 1) reduced ( n = 6). A representative tracing of platelet aggregation is depicted in the lower panel: addition of collagen is indicated by the arrow () and aggregation is reflected by a change of light transmission ( LT) from base line.




Figure 2: Selected ion monitoring of 8-epi PGF. The upper trace shows a peak at m/z 699 corresponding to authentic O-labeled internal standard. The center trace ( m/z 695) shows the signal before the stimulus, and the small peaks are likely to be isoprostanes. The lower trace shows a peak ( m/z 695) corresponding to the retention time of the authentic 8-epi PGF. Other isoprostanes are present, but obscured by the intense 8-epi PGFsignal. The inset shows the trace magnified four times to reveal the other isoprostanes.




Figure 3: Time course of eicosanoid formation in thrombin stimulated washed platelets (0.1-0.3 unit/ml) ( a), and in A23187-stimulated washed platelets (1-3 µM) ( b). Aspirin (asa) 100 µM was incubated for 5 min before the stimulus ( n = 5).



Formation of 8-epi PGFreflected COX activation rather than platelet aggregation. Induction of platelet aggregation in a COX-independent manner by using high doses (10 µg/ml) of collagen in the presence of aspirin was unaccompanied by an increase in 8-epi PGFor TxB(Fig. 4). Similar results were obtained with high dose thrombin. While COX inhibitors suppressed the increment in 8-epi PGFin platelets activated with threshold concentrations of thrombin and collagen, three structurally distinct free radical scavengers, vitamin E, mannitol, and deoxyribose all failed to inhibit its formation (). Aggregation of platelets with threshold concentrations of the thromboxane receptor agonist U46619 (1-3 µM) or of phorbol myristate acetate (100-300 nM) was unassociated with a detectable increment in either TxBor 8-epi PGF.


Figure 4: Aggregation of washed human platelets stimulated with collagen (2 µg/ml) ( A) is associated with 8-epi PGF and TxB formation. Pretreatment with aspirin (100 µM) prevents aggregation ( B) and formation of both. Increasing the concentration of collagen (10 µg/ml) ( C) allows aggregation to occur despite pretreatment with aspirin, but the formation of 8-epi PGFalong with TxBremains inhibited ( n.d., not detectable).



Several lines of evidence are consistent with formation of 8-epi PGFin a COX-dependent manner by human platelets. These include (i) coincident kinetics of formation with the COX product, TxA, in activated platelets; (ii) inhibition of formation coincident with that of TxAby two structurally distinct COX inhibitors; (iii) dissociation of aggregation and 8-epi PGFformation when aggregation occurs in a COX-independent manner; (iv) selective elevation of 8-epi PGFas compared to other isoprostanes.

Effect of Thromboxane Synthase Inhibition on Platelet 8-epi PGFFormation

Washed platelets were incubated with the selective thromboxane synthase inhibitors 1-(7-carboxyheptyl)imidazole hydrochloride and sodium furegrelate (U-63557A) for 5 min at 37 °C, then stimulated with collagen (1 µg/ml) or AA (30 µM). The samples were analyzed for 8-epi PGFand TxBformation. The inhibitors completely prevented TxBproduction, but did not prevent the formation of 8-epi PGFwhich was elevated an average 6-8-fold for both collagen and AA (Fig. 5, A and B) (18) .


Figure 5: Effect of two thromboxane synthase inhibitors, U6557A (50 µM) and carboxyheptyl-imidazole (10 µM), on 8-epi PGF and TxB formation in arachidonic acid ( AA) (30 µM) and collagen ( CL) (1 µg/ml) stimulated washed platelets. The inhibitors were incubated 5 min at 37 °C before adding the stimuli. Pretreatment with 100 µM aspirin ( ASA) abolished aggregation and prevented formation of both TxBand 8-epi PGF. The synthase inhibitors prevented formation of TxBbut an increased production of 8-epi PGFfrom pretreatment values was observed ( n = 5).



Confirmation of 8-Epi PGFas a Product of COX-1 Activity

To verify the authenticity of the endogenous peak comigrating with the internal standard in the quantitative GC-MS assay as 8-epi PGF, we performed several further experiments. First, AA was incubated with partially purified ram seminal vesicle COX-I using the conditions described above. An internal standard, consisting of [O]8-epi PGFwas added to the ether, and the mixture was derivatized as the PFB ester, subjected to TLC, and the tBDMS derivative was formed. When subjected to negative ion chemical ionization GC-MS, a product with the molecular weight and retention time of 8-epi PGFwas observed (not shown).

Second, [H]arachidonic acid was incubated with COX-I and the product extracted by SPE, partially purified by TLC, mixed with 10 µg of authentic 8-epi PGF, and subjected to HPLC purification using the technique of Morrow et al. (5) . 8-Epi PGFwas then monitored by UV absorption at 210 nm; tritiated products were monitored with a Flo-One/Beta radiodetector. Briefly, coelution was demonstrated in three systems: (i) SP HPLC of the underivatized compound, mobile phase: 12% isopropanol, 88% hexane, 0.1% glacial acetic acid, retention time 24.6 min for the unlabeled, and 26 min for the tritiated product; (ii) RP HPLC of the underivatized compound, mobile phase: 28% acetonitrile, 72% water, 0.1% glacial acetic acid, retention time 23.6 min for the unlabeled and 22 min for the tritiated product; and (iii) RP HPLC of the PFB ester, mobile phase: 50% acetonitrile, 50% water, retention time 22.3 min for the unlabeled and 21.9 min for the tritiated product. Retention time differences between the unlabeled material and the products possessing the hydrogen/tritium substitution in eight locations were significant (up to 1.4 min). To account for this variation, the PGFwas purified from the incubation and chromatographed with authentic PGFin the same three systems. The relative retention times enabled compensation for the isotope effect (Fig. 6). The isotope effect was larger than seen by Morrow et al. (5) due to the fact that their standard was labeled with a single tritium.


Figure 6: A) Coelution of a COX-1 product of [H]-arachidonic acid ( - ) with the UV absorption profile of authentic 8-epi PGF (-) after reverse phase HPLC of the underivatized compound (mobile phase: 28% acetonitrile, 72% water, 0.1% glacial acetic acid). The slight difference in retention time reflects the isotope effect (see text). B, coelution of [H]PGF( - ) from the same incubation and authentic PGF(-) using the same HPLC conditions as in A.



Third, 80 µM AA was incubated with washed human platelets and after addition of Ointernal standard, SPE, TLC, and PFB, tBDMS derivatization, the zone cochromatographing with 8-epi PGFwas injected onto a 40 m DB-1 capillary column with a 0.18-mm inner diameter, 0.4-µm coating. Complete GC-MS conditions are described above. This system yielded a retention time of approximately 1 h; no difference in retention time was observed between the [O]8-epi PGFinternal standard and the product of the incubation, other than the expected isotope effect (the internal standard eluted 15 s earlier than both the platelet product and authentic 8-epi PGF).

Gas Chromatography-Electron Impact Mass Spectrometry (GC-EI-MS)

We applied GC-EI-MS to provide structural elucidation of the compound detected in the negative ion chemical ionization GC-MS assay. Platelets were incubated with [5,6,8,9,11,12,14,15-d]arachidonic acid for 3 min at 37 °C. The product of this incubation was subjected to extraction and purification as described above, except that the final derivative was the methyl ester TMS ether and mixed with authentic 8-epi PGF. This technique is useful for demonstrating identity between two compounds because the mass spectrum will be present in two forms that are identical except for the presence of deuterium in one (Fig. 7). For assignment of structure to the ions, we have referred to the study of Middleditch and Desiderio (19) on the mass spectrum of the TMS ether-TMS ester derivative of PGF. Although we used the TMS ether-methyl ester, the spectra are analogous in the ions that contain the ester and, since the ester group plays little role in the fragmentation of most ions, most fragments are identical. The presence of eight deuterons alters the GC retention in a predictable fashion, causing the deuterated analog to elute slightly before the natural product. This difference, although only approximately 1 s, allowed the use of reconstructed ion chromatograms (RIC) to ascertain the origin of each individual ion (see Fig. 8 ). RICs were also useful for demonstrating that several ions present in the spectrum ( m/z 361, 437, and 451) had different GC elution profiles, and therefore did not originate from the compounds of interest.


Figure 7: An electron impact mass spectrum of the product of the incubation of human platelet with [5,6,8,9,11,12,14,15-d]arachidonic acid coinjected with authentic 8-epi PGF derivatized as the methyl ester TMS ether. The lower panel has been magnified by a factor of 3. The inset shows 8-epi PGFmethyl ester TMS ether, the single asterisks (*) denote the sites of deuterium labeling. Ions at m/z 361, 437, and 451 originate from a coeluting impurity (**) (see text).




Figure 8: Reconstructed ion chromatograms showing the slight isotope effect on the GC retention times of the two compounds. m/z 521 and 592 originate from the H-labeled product, as indicated by their slightly earlier (approximately 1 s) retention time.



We interpret the spectrum in the following manner: m/z 584 is the molecular ion of the unlabeled product; its [H] analog is m/z 592. m/z 569 represents the loss of 15 (CH; from TMS); its Hanalog is m/z 577. m/z 513 is the loss of 71 (C16-20); since this portion of the molecule is not labeled, the analog m/z 521 retains all eight deuterons. m/z 494 represents the loss of 90 (trimethylsilanol, TMSOH); the analog can result from a loss of 90 ( m/z 502) or 91 ( m/z 501) depending on whether the loss of TMSOH involves a proton or a deuteron. The ion current is divided between these two ions. m/z 479 is the loss of 105 (90+15; TMSOH+CH). The analog is seen as a loss of 105 ( m/z 487) or 106 ( m/z 486), again depending on whether a proton or deuteron is abstracted. m/z 423 (M-161) originates from the loss of TMSOH (90) and C16-20 (71) . Its analog can occur at M-161 ( m/z 431) or M-162 ( m/z 430) for reasons stated above. m/z 404 reflects the loss of two TMSOH groups (2 90). Since each TMSOH can abstract either a proton or a deuteron, its analog appears at m/z 410, 411, and 412. m/z 397 originates from the exclusion of TMSOCH(116) from M-71. This loss involves C9, with its -OTMS group and C10. Its analog retains seven deuterons and is obscured by the larger m/z 404. An RIC of m/z 404 yields a peak profile consistent with having two components, one originating from the unlabeled standard, the other from the deuterated compound. m/z 333 reflects the loss of 90+90+71. Since its analog can lose zero, one, or two deuterons, it appears at m/z 339, 340, and 341. m/z 307 originates from the expulsion of TMSOCH(116) from M-(90+71). Since C9 with its deuteron is lost and the TMSOH can remove a proton or a deuteron, its analog appears at m/z 313/314. Middleditch and Desiderio (19) described an ion at m/z 313 which could account for the fact that m/z 313 in this spectrum is larger than would be predicted. Two fragments of m/z 243 are reported by Middleditch; the first consists of the intact five-membered ring, the second is C11-15. The former would be expected to retain three deuterons, the latter four. The small ions at m/z 246 and 247 have RICs consistent with their origin from the deuterated product. m/z MDRV 217 originates from a rearrangement of C9-11. It should retain two deuterons, placing its analog at m/z 219. This ion is, in fact, elevated above its expected size, as predicted by other second isotope peaks in the spectrum.

Ex Vivo Study

Basal serum levels for TxB, and 8-epi PGFwere 250 ± 20 ng/ml and 231 ± l5 pg/ml, respectively. Three h after aspirin administration the levels dropped to 4 ± 1 ng/ml and 40 ± 10 pg/ml, respectively, reflecting average reductions of 98% for TxBand 83% for 8-epi PGF. Suppression of serum 8-epi PGF ex vivo persisted 24 h after aspirin administration (Fig. 9).


Figure 9: Serum levels of TxB2 () and 8-epi PGF () before (base) and at 3 and 24 h after administration of aspirin to healthy volunteers ( n = 4).




DISCUSSION

Fisoprostanes are a family of PGFisomers, reportedly formed by free radical catalyzed peroxidation of arachidonic acid, independent of the action of COX (1) . Evidence for this pathway of formation includes enhanced formation in vivo in animal models of enhanced oxidative stress, such as poisoning with carbon tetrachloride (20) , or depletion of endogenous antioxidants, such as by iron overload (21) . Isoprostanes are formed initially from arachidonic acid in situ in phospholipids and are putatively cleaved from the membrane by phospholipases A(22, 23) .

Following hepatic injury with carbon tetrachloride, formation in the membrane precedes their release into the effluent when the liver is perfused in situ. Similarly, oxidation of low density lipoprotein, either by Cu, a metal-independent source of peroxyl radicals, or by coincubation with endothelial cells (24, 25, 26) results in initial formation in the phospholipid, followed by release of the isoprostanes. Although the precise chemistry of the reactions which lead to their formation remains to be elucidated, the discovery of isoprostanes affords a potential index of free radical catalyzed events in vivo, as they can be measured in plasma and urine (12, 27) .

Attention has particularly focused upon one of these compounds, 8-epi PGF, which has been shown to possess biological activity as a vasoconstrictor (6, 7) . This effect is prevented by pharmacological antagonists of the thromboxane receptor. It is unclear whether 8-epi PGFincidentally exerts its effects via this receptor or whether it acts via a distinct, but related receptor (8) . Pharmacological assessment of the effects of 8-epi PGFsuggests that it acts as a partial agonist at the thromboxane receptor that transduces the aggregation response (10, 12, 28) . Interestingly, despite its lack of potency as a stimulant of aggregation, 8-epi PGFcan readily elicit the platelet shape change that normally precedes the aggregation response, and this phenomenon is accompanied by an increase in [Cai] (12, 28) . We have previously shown that the thromboxane antagonist, GR 32191, segregates forms of the thromboxane receptor which mediate aggregation and the release reaction from those which transduce the platelet shape change and vasoconstriction (29, 30) .

To elucidate the mechanism of formation of Fisoprostanes in human platelets, we initially studied the time course of appearance of 8-epi PGFin response to activation by the physiological agonists collagen and thrombin. We developed a stable isotope dilution assay, using an O-labeled internal standard and gas chromatography-mass spectrometry (11) . To our surprise, there was a marked increase in the peak comigrating with the internal standard for 8-epi PGF, coincident with platelet aggregation. Importantly, closely migrating peaks, presumed to reflect other Fisoprostanes, did not increase correspondingly. Pretreatment with the COX inhibitors aspirin and indomethacin completely abolished this increment in 8-epi PGF, together with the major COX product in platelets, thromboxane A, as reflected by its hydrolysis product, thromboxane B. Furthermore, induction of aggregation in a COX-independent manner by a variety of approaches (the phorbol ester phorbol 12-myristate 13-acetate, the endoperoxide analog U46619, or by high doses of collagen or thrombin in the presence of aspirin) was unassociated with 8-epi PGFformation.

The possibility existed that the peak in the endogenous material corresponding to the internal standard for 8-epi PGFactually represented several unresolved species. To address this possibility, we obtained several lines of evidence that the COX metabolite was actually 8-epi PGF. First, we added radiolabeled arachidonic acid to the semipurified COX. The tritiated product of this reaction was then mixed with authentic 8-epi PGFand passed over three highly resolving chromatographic systems; the product of the reaction coelutes with authentic 8-epi PGF.

More evidence was acquired by obtaining an electron impact mass spectrum of a mixture of authentic 8-epi PGFand the product isolated from an incubation of octadeuterated AA with platelets. The mass spectrum shows a series of ions corresponding to those of 8-epi PGFand another series corresponding to a deuterated analog. While the EI mass spectrum of PGFwould be similar, this compound is clearly chromatographically resolved from 8-epi PGFunder the conditions of the assay.

Third, human platelets made octadeuterated 8-epi PGFfrom octadeuterated arachidonic acid in an aspirin-sensitive manner, as detected in a selected ion monitoring assay. If the 8-epi PGFhad been formed from either PGDor PGE, this would have involved loss of one of the ring deuteriums, resulting in a heptadeuterated product (31) . Thus, human platelets and perhaps other cells, possess the capacity to form 8-epi PGFas a prostaglandin.

Data consistent with this observation have been previously reported by others. Corey et al. (32) proposed a schema by which 8-epi PGFmight be formed via the corresponding endoperoxide by a biomimetic cyclization and Hecker et al. (9) demonstrated that 8-epi PGFis a product of the ram seminal vesicle COX. Additional support for a COX-dependent source of 8-epi PGFformation in human platelets is provided by two experiments with structurally distinct inhibitors of thromboxane synthase. These compounds increase 8-epi PGFformation coincident with inhibition of TxA(18) . Finally, we have shown coelution of the putative COX-1 product with authentic 8-epi PGFby GC-MS as the PFB tBDMS derivative and as the ME-TMS derivative. We have also shown HPLC coelution of the underivatized compounds by straight phase chromatography and of the PFB ester derivative by straight phase and reverse phase chromatography.

The formation of 8-epi PGFin a COX-dependent manner does not exclude the possibility that this reflects generation of free radicals associated with enzyme turnover. However, formation via peroxyl radical isomers of arachidonate is unlikely. We found that three structurally distinct, free radical scavengers failed to prevent 8-epi PGFformation by stimulated human platelets under conditions where aspirin was effective. Furthermore, its production was not accompanied by the appearance of the array of isomers typical of isoprostane production. Finally, the biomimetic schema outlined by Corey et al. (32) renders such an explanation unnecessary.

Formation of the compound as a prostaglandin may provide intuitive support for the concept of distinct receptors for 8-epi PGF, as has been suggested (8) . Although its biological effects are prevented by thromboxane receptor antagonists, it displaces ligand weakly (8, 12) from the recombinant thromboxane receptor cloned from human placenta (33) . The recombinant PGFreceptor is not activated by 8-epi PGF.() A single thromboxane receptor gene has been cloned (34) . However, subtle changes in primary sequence may result in discriminant affinity for ligands (35) , and the recent discovery of tissue specific expression of splice variants (36) raises the possibility that 8-epi PGFmight exhibit greater affinity for a variant other than the placental isoform. Alternatively, post-translational modifications of the receptor, perhaps initiated by oxidizing conditions, may favor 8-epi PGFas a ligand.

The biological importance of this compound as an autacoid remains to be established. It is noteworthy in this regard, that 8-epi PGFis a considerably less abundant COX product than thromboxane in human platelets. However, the demonstration that 8-epi PGFis formed in serum and that it is substantially depressed ex vivo in volunteers administered aspirin indicates the potential relevance of these observations to its formation in vivo. Several of the clinical situations putatively associated with free radical generation, in which we have found elevated urinary 8-epi PGF, including syndromes of vascular reperfusion and chronic cigarette smoking, are associated with platelet COX activation (13, 14) . Increased excretion of 8-epi PGFmay reflect the generation of free radicals by the vasculature (37) in these clinical settings. If so, utilization of such an index would be pertinent to the rational development of antioxidant drugs in humans. Clearly, delineation of the extent to which a COX-dependent pathway might confound interpretation of urinary 8-epi PGFis critical to its use as a quantitative index of free radical generation in vivo.

  
Table: Effect of aspirin (ASA) and the antioxidants, vitamin E (vit E, 500 µM), mannitol (MN, 5 mM) and deoxyribose (DR, 5 mM) on eicosanoid formation in collagen and thrombin-stimulated washed platelets ( n = 5).

The antioxidants and aspirin were incubated for 5 min at 37 °C before adding the stimuli.



FOOTNOTES

*
This work was supported by a program grant from the Wellcome Trust and the European Union (to G. A. F.) and FATMA Project Grant 82.00004.41 from the Italian National Research Council (to D. P.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked `` advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Center for Experimental Therapeutics, 909 Biomedical Research Bldg., 422 Curie Blvd., University of Pennsylvania, Philadelphia, Pa. 19104. Fax: 215-573-9004.

The abbreviations used are: PG, prostaglandin; COX, cyclooxygenase; Tx, thromboxane; SPE, solid-phase extraction; TLC, thin layer chromatography; HPLC, high performance liquid chromatography; GC, gas chromatography; MS, mass spectrometry; EI, electron impact; SP, straight phase; RP, reverse phase; PFB, pentafluorobenzyl; ASA, aspirin; 12-HETE, 12-hydroxyeicosatetraenoic acid; PRP, platelet-rich plasma; PPP, platelet-poor plasma; WP, washed platelets; TC, threshold concentration; CL, collagen; AA, arachidonic acid; U46619, 9,11-dideoxy-9a,11a-methanoepoxy PGF; RIC, reconstructed ion chromatogram; BSTFA, bis-[trimethylsilyl]trifluoroacetamide; MTBSTFA, N-[ tert-butyldimethylsilyl]- N-methyltrifluoroacetamide; ODS, octadecylsilyl; TMS, trimethylsilyl.

A. Ford-Hutchinson, personal communication.


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