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Journal of Lipid Research, Vol. 43, 495-509, March 2002
Copyright © 2002 by Lipid Research, Inc.

Methods

Identification of metabolites from Type III F₂-isoprostane diastereoisomers by mass spectrometry

Correspondence to: Chiara Chiabrando, To whom correspondence should be addressed., chiabrando{at}marionegri.it (E-mail)

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

F₂-isoprostanes (F₂-iPs) are prostaglandin (PG)-like products of non-enzymatic free radical-catalyzed peroxidation of arachidonic acid that are now widely used as indices of lipid peroxidation in vivo. Knowledge of the metabolic fate of F₂-iPs in vivo is still scant, despite its importance for defining their overall formation and biological effects in vivo. Type III F₂-iPs, which are diastereoisomers of cyclooxygenase-derived PGF₂, may be metabolized through the pathways of PG metabolism. We therefore studied the in vitro metabolism of eight synthetic Type III F₂-iP diastereoisomers in comparison with PGF₂. We used gas chromatography-mass spectrometry and high performance liquid chromatography-electrospray-tandem mass spectrometry for structural identification of metabolites formed after incubation of the various compounds with isolated rat hepatocytes. PGF₂ was metabolized to several known products, resulting from a combination of ß-oxidation, reduction of ⁵ and/or ¹³ double bonds, and 15-OH oxidation, plus other novel products deriving from conjugation with taurine of PGF₂ and its metabolites. Of the eight F₂-iP diastereoisomers, some were processed similarly to PGF₂, whereas others showed peculiar metabolic profiles according to specific stereochemical configurations.

These data represent the first evidence of biodegradation of selected Type III F₂-iP isomers other than 8-epi-PGF₂, through known and novel pathways of PGF₂ metabolism. The analytical characterization of these products may serve as a basis for identifying the most significant products formed in vivo. — Chiabrando, C., C. Rivalta, R. Bagnati, A. Valagussa, T. Durand, A. Guy, P. Villa, J-C. Rossi, and R. Fanelli. Identification of metabolites from Type III F₂-isoprostane diastereoisomers by mass spectrometry. J. Lipid Res. 2002. 43: 495–509.

Supplementary key words: PGF₂, taurine conjugation, isolated rat hepatocytes, gas chromatography-mass spectrometry, high performance liquid chromatography-electrospray-tandem mass spectrometry

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

F₂-isoprostanes (F₂-iPs), a complex family of 64 compounds isomeric to cyclooxygenase-derived PGF₂, are products of non-enzymatic free radical-catalyzed peroxidation of arachidonic acid (1) (2) (3). Because these compounds are now widely used as indices of lipid peroxidation in vivo (2) (3) (4) (5), knowledge of their metabolic fate may be useful, in general, to better define their overall formation. In fact, the identification of major metabolites of F₂-iPs might help in finding new analytical targets to monitor in addition to, or instead of, the parent compounds. Moreover, given that selected F₂-iP isomers have potent biological effects (2) (3) (4) (5), it is important to establish whether and how these isomers are specifically degraded, to relate their levels in vivo to a putative biological effect.

Depending on the position of the free radical-initiated hydrogen abstraction, four classes of F₂-iP regioisomers can theoretically be formed by peroxidation of arachidonic acid. These families were first called Type I, II, III, and IV (1), but are now known as 5-, 12-, 8-, and 15-Series (2), or Type VI, V, IV, and III (6). Using the latter nomenclature (6), removal of a hydrogen at C-10 gives rise to the formation of two regioisomer families (Type IV and Type V), whereas at C-7 or C-13, it leads to the Type VI or Type III family, respectively. Regioisomer families of Type VI and Type III, theoretically predicted as the most abundant, did indeed predominate over Type V and IV in vivo (1) (6) (7).

F₂-iPs of Type III are of particular interest because they are diastereoisomers of cyclooxygenase-derived PGF₂ ( Fig 1), and may therefore mimic some of the biological properties of PGF₂ or other prostanoids. Although the biological effects of most F₂-iP isomers are largely unknown, 8-epi-PGF₂ and 12-epi-PGF₂ do indeed have biological effects that appear to be mediated by prostanoid receptors (TP and/or FP) (2) (3) (4) (5). In view of their structural similarity with PGF₂, Type III F₂-iPs may also interact with the enzymes that degrade prostaglandins in vivo, but it is difficult to predict the influence of the different steric configurations on the various metabolic pathways.

View larger version (18K): [in this window] [in a new window]   Figure 1. Structures of PGF2 and 8,12-cis-9,11-cis Type III F2-iPs.

To date, the only F₂-iP metabolites identified as products of selected isomers are metabolites of 8-epi-PGF₂, the best-known and most-studied isomer (2) (3) (4) (5). 2,3-Dinor-5,6-dihydro-8-epi-PGF₂ was reported as the only noteworthy metabolite of 8-epi-PGF₂ in humans (8). We have, however, recently shown that at least two major metabolites deriving from endogenous 8-epi-PGF₂ exist in human and rat urine (2,3-dinor-5,6-dihydro-8-epi-PGF₂ and 2,3-dinor-8-epi-PGF₂) (9). We also found that these same metabolites, in addition to 2,3,4,5-tetranor-8-epi-PGF₂, are formed when 8-epi-PGF₂ is incubated with isolated rat hepatocytes (9). In the rabbit, 13,14-dihydro-15-keto-2,3,4,5-tetranor-8-epi-PGF₂ was identified as the most abundant urinary metabolite of exogenous 8-epi-PGF₂ (10).

As we showed recently (9), searching for unknown endogenous metabolites of F₂-iPs is very complex, because of the simultaneous presence in vivo of a myriad of isomeric compounds. In fact, to obtain stereo- and enantio-specific identification of selected products, special analytical approaches have to be devised ad hoc (9). To obtain a general indication, as a first step, about which metabolites can be expected in vivo, we studied in vitro the metabolic fates of PGF₂ and Type III F₂-iPs. Among the 16 diastereoisomers of this family, we studied the eight with cis ring substituents, which are believed to be the most abundant (6) (Fig 1). The different diastereoisomers were incubated with isolated rat hepatocytes, and their metabolites identified by gas chromatography-negative ion chemical ionization mass spectrometry (GC-NICIMS) and high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry (HPLC-MS/MS).

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Materials
8-Epi-PGF₂, 3,3',4,4'-[²H₄]-8-epi-PGF₂, and 2,3-dinor-5,6-dihydro-8-epi-PGF₂ were purchased from Cayman Chemical Co. (Ann Arbor, MI). PGF₂ and 13,14-dihydro-15-keto-PGF₂ were from Upjohn Co. (Kalamazoo, MI). 15R-8-epi-PGF₂, ent-8-epi-PGF₂ (15R and 15S), 12-epi-PGF₂ (15S and 15R), ent-12-epi-PGF₂ (15R and 15S), 2,3-dinor-8-epi-PGF₂, 2,3-dinor-ent-8-epi-PGF₂, 2,3-dinor-5,6-dihydro-ent-8-epi-PGF₂, and 13,14-dihydro-15-keto-2, 3-dinor-5,6-dihydro-ent-8-epi-PGF₂ were synthesized in our laboratories (11) (12) (13).

Isolated rat hepatocytes
Hepatocytes were isolated as described previously (9) from fed male Crl:CD (SD) BR rats.³ Procedures involving animals and their care were conducted in conformity with the institutional guidelines that are in compliance with national (D.L. n. 116, G.U., suppl. 40, 18 Febbraio 1992, Circolare No. 8, G.U., 14 Luglio 1994) and international laws and policies (EEC Council Directives 86/609, OJ L 358, 1, Dec. 12, 1987; Guide for the Care and Use for Laboratory Animals, U.S. National Research Council, 1996).³ Briefly, hepatocytes obtained after perfusing the liver with a collagenase solution were washed and suspended in Leibovitz L-15 medium (Gibco, Scotland) supplemented with HEPES 18 mM, insulin (1 µg/ml), gentamicin (50 µg/ml), and 5% FCS (Gibco) at a density of 1 x 10⁶ cells/ml. After 3 h adhesion the medium was replaced, but without FCS and gentamicin.

Incubation of PGF₂ and related F₂-iPs
Plates were prepared with 3 x 10⁶ cells. The incubation medium was enriched with 670 ng/ml of PGF₂, 8-epi-PGF₂, or the other F₂-iPs under test. In a preliminary experiment to test the experimental conditions, cells were incubated for 10, 20, and 40 min with or without PGF₂ and 8-epi-PGF₂. In the other experiments, PGF₂ and the various F₂-iPs were incubated separately for 20 min. At the end of the incubation, the medium was collected and spun at 4°C to eliminate non-adhering hepatocytes. Cell-free medium ad plates were stored at -20°C until analyzed.

Extraction
Metabolites were extracted from the incubation medium (2.5 ml) after addition of [²H₄]-8-epi-PGF₂ and [²H₄]-PGF₂ (50 ng each) as internal standards. The medium was diluted to 10 ml with 1 mM HCl, acidified to pH 3, and extracted on C18 solid phase extraction columns (SPE; Bakerbond) preconditioned with methanol and 1 mM HCl. After two washing steps (water and petroleum ether, 10 ml each), the columns were eluted with 2 ml methyl formate (eluate A) and then 2 ml methanol (eluate B). After drying, eluates were assayed separately as follows. Eluate A (containing the unmetabolized parent compounds and unconjugated metabolites), was derivatized (see below) and analyzed by GC-NICIMS, or reconstituted in the HPLC initial mobile phase (see below) and directly analyzed by HPLC-MS/MS. Eluate B (containing conjugated metabolites), was analyzed by HPLC-MS/MS. Metabolites were also extracted from plate-adhering hepatocytes by 1) adding ethanol and internal standards to plates (4°C overnight), 2) suspending the cell lysate, 3) vigorously vortexing, centrifuging and drying the ethanol suspension, and 4) resuspending the residue in 1 mM HCl and extracting as above. In experiments to measure the unmetabolized parent compounds, 0.025 ml medium and 5 ng internal standards were used.

Derivatization
Samples were converted to pentafluorobenzyl ester (PFB), methoxime (MO), and trimethylsilyl ethers (TMS), or PFB-TMS as follows: 1) add 30 µl pentaflourobenzyl bromide-acetonitrile (1:5, v/v) and 5 µl diisopropylethylamine to the dry residue (40°C for 5 min); 2) repeat previous step; 3) add 50 µl methoxyamine HCl in pyridine (Pierce, Rockford, IL), (60°C for 1 h); 4) dry; and 5) add 50 µl N,O-bis-(trimethylsilyl)trifluoroacetamide (Fluka, Buchs, Switzerland) (60°C for 1 h).

GC-NICIMS
Analyses were as described (9), using a Finnigan 4000 mass spectrometer. Briefly, GC operating conditions were: NB-54 (5% phenyl, 95% dimethylpolysiloxane) fused silica capillary column (25 m length, 0.32 mm ID, 0.12 µm film thickness); solvent-split injection (50°C to 300°C); oven temperature, isothermal at 160°C for 1 min, then programmed to 300°C at 10°/min; helium as carrier gas. Negative ion chemical ionization (NICI) operating conditions were: selected ion recording (SIR) of carboxylate anions (M-181, loss of PFB); methane as reagent gas; electron energy, 100 eV.

HPLC-MS/MS
Analyses were done on an API 3000 triple quadrupole instrument (PE Sciex, Thornhill, Ontario, Canada), interfaced with two Series 200 micro liquid chromatography (LC) pumps (Perkin-Elmer, Norwalk, CT). HPLC conditions were as follows: column, Superspher C_18e, 3.5 µm, 2 x 125 mm (Merck, Darmstadt, Germany); flow, 0.2 ml/min; eluent A, 0.1% formic acid; eluent B, acetonitrile; gradient, from 10% to 52% B in 28 min; loop, 20 µl. The HPLC effluent was directed to the MS instrument through a splitting T-connection, reducing the flow into the ion source to about 40 µl/min. MS conditions were as follows: source, TurboIonSpray in negative ion mode; heater gas, 280°C; ion spray voltage, -4,000 V; orifice voltage, -56 V; ring voltage, -250 V; collision cell gas, nitrogen at a pressure of 2.6 x 10^-5 torr; collision energy, 26 to 56 eV (see Results). To optimize ionization and Q2 decomposition, authentic standards were directly infused into the ion source, or MS/MS loop experiments with different collision energies were run on the HPLC-eluted biological material.

Metabolite quantitation
To compare the metabolic profiles of unconjugated metabolites of the various diastereoisomers, we assayed the hepatocyte medium by GC-NICIMS in the SIR mode. In these experiments, ²H₄-8-epi-PGF₂ (5 ng) was added to 0.25 ml of incubation medium before C18 solid phase extraction (SPE). Quantitation was done by comparing peak area ratios of the carboxylate anion of each metabolite to that of the internal standard, using for reference the calibration curve of 8-epi-PGF₂/²H₄-8-epi-PGF₂ (9). This was done assuming that all metabolites gave an approximately similar molar response on the NICIMS recording of their respective carboxylate anions (9). For compounds bearing the 15-keto group, giving syn/anti isomers of the methoxime derivative, the total peak area was used for calculation.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Identification strategy for PGF₂ and F₂-iPs
The hepatocyte incubation medium was first screened for the presence of major metabolites of PGF₂ and 8-epi-PGF₂ by GC-NICIMS. We based ourselves on earlier descriptions of PGF₂ metabolism in various species (14) (15) (16) (17) (18) (19) and our previous characterization of ß-oxidation products of PGF₂ and 8-epi-PGF₂ (9), and used full scan and SIR to screen for the carboxylate anion clusters of the PFB esters of metabolites possibly formed from the nine diastereoisomers. Fig 2 shows a representative SIR tracing of the unconjugated metabolites of 8-epi-PGF₂.

View larger version (37K): [in this window] [in a new window]   Figure 2. Gas chromatography-negative ion chemical ionization mass spectrometry (GC-NICIMS) selected ion recording tracing of carboxylate anions (M-PFB)- of 8-epi-PGF2 and its unconjugated metabolites formed by isolated rat hepatocytes. The compounds were extracted from the incubation medium and derivatized to pentafluorobenzyl ester/methoxime/trimethylsilyl ether (PFB/MO/TMS). 13,14DH-15K, 13,14-dihydro-15-keto; 2,3-dinorDH, 2,3-dinor-5,6-dihydro. The peak labeled with an asterisk is from the isotopic cluster of 2,3-dinor metabolite.

The unconjugated products tentatively identified by this screening method were then structurally identified after HPLC separation by examining the mass spectra of the product ions formed after collision-activated decomposition (CAD) of the carboxylate anions produced by electrospray ionization (HPLC-MS/MS) ( Table 1). Fig 3 shows a representative HPLC-MS/MS tracing of the unconjugated metabolites of 8-epi-PGF₂.

View larger version (23K): [in this window] [in a new window]   Figure 3. High-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) tracing of total ion current (TIC) of product ions generated by collision-activated decomposition of carboxylate anions (M-H)- of 8-epi-PGF2 and its unconjugated metabolites formed by isolated rat hepatocytes. The compounds were extracted from the incubation medium. 13,14DH-15K, 13,14-dihydro-15-keto; 2,3-dinorDH, 2,3-dinor-5,6-dihydro.

View this table: [in this window] [in a new window]   Table 1. Metabolites of PGF2 and F2-iPs identifieda in isolated rat hepatocyte incubation media

Chromatographic and mass spectral features of parent compounds, their metabolites, and authentic standards are summarized in Table 2 (GC-NICIMS) and Table 3 (HPLC-MS/MS). PGF₂ and its two epimers at C-8 (8-epi-PGF₂) and C-12 (12-epi-PGF₂) gave excellent separation by both GC and HPLC, and their corresponding epimeric metabolites behaved similarly (Table 2 and Table 3). For all compounds, the steric configuration of the hydroxyl at C-15 had little or no influence on the retention time in both techniques in the conditions used (Table 2 and Table 3). The enantiomeric pairs (e.g., 8-epi-PGF₂ and ent-8-epi-PGF₂) showed the expected identical behavior, since they obviously need a chiral separation step to be resolved (Table 2 and Table 3). All diastereoisomers gave identical MS/MS spectra, as shown for example for PGF₂ and 8-epi-PGF₂ ( Fig 4), as reported by Waugh et al. (7).

View larger version (25K): [in this window] [in a new window]   Figure 4. Tandem mass spectrometry (MS/MS) spectra obtained from collision-activated decomposition of the carboxylate anions (m/z 353; 38 eV) of parent PGF2 and 8-epi-PGF2, plus authentic 13,14-dihydro-15-keto-PGF2 and hepatocyte-derived 13,14-dihydro-15-keto-8-epi-PGF2. HPLC retention time is indicated for each metabolite.

View this table: [in this window] [in a new window]   Table 2. GC-NICIMS behavior of PGF2, Class III F2-iPs and their unconjugated metabolites as PFB-TMS or PFB-MO-TMSa derivatives

View this table: [in this window] [in a new window]   Table 3. HPLC-MS/MS behavior of PGF2, Class III F2-iPs and their unconjugated metabolites

Most unconjugated compounds were identified by direct comparison with the corresponding standard synthesized ad hoc in our laboratories (Table 2, Table 3, and Table 4). Metabolites lacking the corresponding reference compounds were identified by comparison with an authentic diastereoisomeric analog (Table 2 and Table 3). Additional identification criteria are described below in the specific sections. MS/MS spectra of selected diastereoisomeric unconjugated metabolites are shown in Fig 4, Fig 5 and Fig 6.

View larger version (23K): [in this window] [in a new window]   Figure 5. MS/MS spectra obtained from collision-activated decomposition of the carboxylate anions of authentic 2,3-dinor-8-epi-PGF2 and hepatocyte-derived 2,3-dinor-8-epi-PGF2 (m/z 325; 22 eV), and hepatocyte-derived 2,3,4,5-tetranor-8-epi-PGF2 and 13,14-dihydro-15-keto-2,3,4,5-tetranor-8-epi-PGF2 (m/z 299; 30 eV). HPLC retention time is indicated for each metabolite.

View larger version (28K): [in this window] [in a new window]   Figure 6. MS/MS spectra obtained from collision-activated decomposition of the carboxylate anions (m/z 327; 40 eV) of authentic 2,3-dinor-5,6-dihydro-8-epi-PGF2, hepatocyte-derived 2,3-dinor-5,6-dihydro-PGF2, authentic 13,14-dihydro-15-keto-2,3-dinor-5,6-dihydro-ent-8-epi-PGF2, and hepatocyte-derived 13,14-dihydro-15-keto-2,3-dinor-5,6-dihydro-PGF2. HPLC retention time is indicated for each metabolite.

View this table: [in this window] [in a new window]   Table 4. Product ionsa detected by MS/MS after fragmentation of carboxylate anions [M-H]- of PGF2, Class III F2-iPs and their unconjugated metabolites

For simplicity, the various diastereoisomeric metabolites identified for PGF₂ and F₂-iPs are described by referring to their common chemical structure. Identification criteria for conjugated metabolites are reported below.

Metabolites of PGF₂ and Type III F₂-iPs
Metabolism of PGF₂ by isolated rat hepatocytes was studied as a reference for the eight 8,12-cis-9,11-cis Type III F₂-iP diastereoisomers (Fig 1). Some of the compounds identified here as PGF₂ metabolites are products of known metabolic pathways, i.e., one or two steps of ß-oxidation, reduction of ⁵ and/or ¹³ double bonds, and 15-OH dehydrogenation ( Fig 7). We also identified novel taurine conjugates of PGF₂ and its metabolites. Some, but not all, of these compounds were also formed from F₂-iPs, which were in fact metabolized according to stereoselective patterns (Table 1).

View larger version (17K): [in this window] [in a new window]   Figure 7. Structures of unconjugated metabolites identified in this study. Formation of taurine conjugates at C1 (taurine-conj) is also indicated (structures shown in Fig 8).

The overall view of the metabolites identified for the nine PGF₂ diastereoisomers (Table 1) shows that: 1) all metabolites of PGF₂ were also present as products of some, although not all, F₂-iPs; 2) some metabolites were exclusive products of F₂-iPs (i.e., all 2,3-dinor metabolites with intact ⁵ double bond); 3) among F₂-iPs, 8-epi-PGF₂ had the largest number of products in common with PGF₂; 4) the only products common to all nine diastereoisomers were the 2,3,4,5-tetranor metabolites and the taurine conjugates of the parent compounds; 5) within the 8-epi series, those with the 15R configuration were not transformed into any of the seven possible metabolites bearing the 13,14-dihydro-15-keto modification; and 6) within the 12-epi series, each enantiomeric pair showed a characteristic profile.

Unconjugated metabolites To help interpret the MS/MS spectra of the metabolites, the product ions formed from the precursor carboxylate anions of the metabolites are listed in Table 4 in comparison with those of parent PGF₂ or F₂-iPs, plus 3,3',4,4'-²H₄-8-epi-PGF₂, and 1,1'-¹⁸O₂-2,3-dinor-5,6-dihydro-8-epi- PGF₂. All mass spectral data refer to authentic standards and corresponding metabolites, except for hepatocyte-derived 13,14-dihydro-15-keto-2,3-dinor metabolite and the two tetranor compounds. The structural interpretation given in Table 4 is mainly based on work by Murphy's group (7) (20) (21), or deducted from the comparison of the various metabolites and stable isotope-labeled analogs. Product ions of the carboxylate anion of 8-epi-PGF₂ or PGF₂, that reportedly comprise the -chain, had the expected counterparts in the -chain shortened metabolites, with shifts of 28 amu less (2,3-dinor), 26 amu less (2,3-dinor-5,6-dihydro), and 54 amu less (2,3,4,5-tetranor) (Table 4). This and other specific features described below allowed identification of the three compounds for which no authentic standard was available.

2,3-dinor metabolite. No compound with a 2,3-dinor modification and intact ⁵ double bond was found for PGF₂, confirming previous observations in the rat in vitro and/or in vivo by ourselves (9) and others (16) (17) (18). The 2,3-dinor metabolite was, however, identified as a prominent product of several F₂-iP diastereoisomers, namely 8-epi-PGF₂, 15R-8-epi-PGF₂, ent-8-epi-PGF₂, 15S-ent-8-epi-PGF₂, 12-epi-PGF₂, and ent-12-epi-PGF₂ (Table 1, Table 2, Table 3). 2,3-Dinor-8-epi-PGF₂, which we had characterized previously as a prominent endogenous metabolite in human and rat urine, as well as a major product of 8-epi-PGF₂ metabolism by rat hepatocytes, was now compared with authentic 2,3-dinor-8-epi-PGF₂ recently synthesized in our laboratories (Fig 5). While the carboxylate anions of parent compounds and other metabolites produced several fragment ions mainly due to single and combined losses of H₂O, CO₂, and C₂H₄O from the ring, and various parts of the alkyl chains (7) (20) (21), the carboxylate anion of the 2,3-dinor metabolite (m/z 325) gave a peculiar fragmentation (Table 4 and Fig 5). Even at a low collision energy (22 eV), its MS/MS spectrum was dominated by a main fragment ion at m/z 237, probably due to the loss of C₂H₄O plus CO₂ from the carboxyl. Other minor ions were observed at m/z 263 (loss of H₂O and C₂H₄O), m/z 219 (loss of H₂O, C₂H₄O and CO₂), m/z 163 (loss of H₂O, CO₂ and hexanal), and m/z 137. Similar MS/MS spectra (not shown) were recorded for the corresponding metabolites derived from the other F₂-iPs.

2,3-dinor-5,6-dihydro metabolite. This was a prominent product of PGF₂, 12-epi-PGF₂, and ent-12-epi-PGF₂, and a minor product of 8-epi-PGF₂ and ent-8-epi-PGF₂ (Table 1 Table 2 Table 3). These compounds were identified by direct comparison with commercial 2,3-dinor-5,6-dihydro-8-epi-PGF₂ and synthetic 2,3-dinor-5,6-dihydro-ent-8-epi-PGF₂. Fig 6 shows the MS/MS spectra of authentic 2,3-dinor-5,6-dihydro-8-epi-PGF₂ and hepatocyte-derived 2,3-dinor-5,6-dihydro-PGF₂, as an example. The precursor ion (m/z 327) was fragmented according to the typical pattern of the PGF₂ compounds (Table 4). The MS/MS spectrum of 2,3-dinor-5,6-dihydro-8-epi-PGF₂ labeled with ¹⁸O₂ enabled us to confirm the proposed fragmentation by observing the expected shift of 4 amu in all fragment ions conserving the carboxyl (Table 4). However, the ion at m/z 167 did not shift to m/z 171, as expected, when the carboxyl was labeled with ¹⁸O₂. The putatively corresponding ion of 8-epi-PGF₂ at m/z 193 shifts in fact to m/z 197 upon labeling with ¹⁸O₂, indicating retention of the carboxyl (22). Our data indicate that for 2,3-dinor-5,6-dihydro-8-epi-PGF₂, this fragment is instead due to the loss of CO₂, and not C₂H₄O, in addition to part of the -chain.

2,3,4,5-tetranor metabolite. A metabolite with a 2,3,4,5-tetranor structure, resulting from two ß-oxidation steps, was common to PGF₂ and all F₂-iPs (Table 1), indicating that this pathway [in addition to conjugation with taurine (see below)] is the least affected by the stereochemical structure of the parent compound. This was a prominent product for all diastereoisomers, except 15S-ent-8-epi-PGF₂, which had a much more abundant 13,14-dihydro-15-keto-2,3,4,5-tetranor metabolite (see below). Our previous characterization of the 2,3,4,5-tetranor metabolites of PGF₂ and 8-epi-PGF₂ (9) helped prove the identity of the other diastereoisomeric metabolites. The chromatographic and mass spectral characteristics of the nine diastereoisomeric 2,3,4,5-tetranor metabolites (Table 2 and Table 3) clearly support their identity. The MS/MS spectra of the carboxylate anion (m/z 299) showed a fragmentation consistent with the proposed structure (Table 4 and Fig 5). All ions conserving the -chain were found, as expected, at a m/z value 54 amu less than the corresponding ions of the parent compound.

13,14-dihydro-15-keto metabolite. A metabolite with this structure was found for PGF₂ and for two of the F₂-iPs, namely 8-epi-PGF₂ and 15S-ent-8-epi-PGF₂ (Table 1). The product deriving from PGF₂ had GC-NICIMS and HPLC-MS/MS behavior identical to authentic 13,14-dihydro-15-keto-PGF₂ (Table 2 and Table 3, and Fig 4). On GC, the two syn/anti isomers due to derivatization of the keto group to methoxime eluted shortly after the parent compounds, as a single broad peak for 13,14-dihydro-15-keto-PGF₂ but as separate peaks for the metabolites from 8-epi-PGF₂ and 15S-ent-8-epi-PGF₂ (Table 2 and Fig 2). On HPLC, the 13,14-dihydro-15-keto diastereoisomeric metabolites eluted about 3 min later than their parent compounds (Table 3 and Fig 3). The MS/MS spectra of hepatocyte-derived and authentic 13,14-dihydro-15-keto-PGF₂ are shown in Fig 4. On account of their identical molecular weight, the 13,14-dihydro-15-keto metabolite and the parent compound have the same mass-to-charge ratio for the carboxylate anion and several product ions (Table 4). The MS/MS spectrum of the 13,14-dihydro-15-keto metabolite can, however, be clearly distinguished by the prominent ions at m/z 113, 183, and 195, and the very low abundance of m/z 193 (base peak in the parent compound). The corresponding metabolites from 8-epi-PGF₂ and 15S-ent-8-epi-PGF₂ had identical MS/MS spectra (not shown).

13,14-dihydro-15-keto-2,3-dinor metabolite. A compound with this structure was found only as a minor product of 8-epi-PGF₂ (Table 1). This metabolite was identified by observing its GC-NICIMS and HPLC-MS/MS characteristics, which are consistent with its putative structure. In particular, GC-NICIMS analysis showed the peak doublet of the syn/anti isomers (RT = 10.7 and 10.8 min) at m/z 498 when derivatized to methoxime, but a single peak (RT = 10.9 min) at m/z 469 (29 amu less, corresponding to the different groups at C-15, i.e., C=NOCH₃ vs. C=O), when the keto group was not derivatized (not shown). The MS/MS spectrum, although poorly informative, was very similar to that of the corresponding metabolite without the 13,14-dihydro-15-keto modification (Table 4), except for a difference in the most abundant ion (base peak at m/z 263).

13,14-dihydro-15-keto-2,3-dinor-5,6-dihydro metabolite. A compound with this structure was found only as a minor product of PGF₂ and 8-epi-PGF₂ (Table 1). This metabolite was identified by direct comparison with authentic 13,14-dihydro-15-keto-2,3-dinor-5,6-dihydro-ent-8-epi-PGF₂ using both GC-NICIMS and HPLC-MS/MS (Table 2 and Table 3). The MS/MS spectra of the standard and the metabolite from PGF₂ are shown in Fig 6. The mass spectrum was similar for the corresponding metabolite derived from 8-epi-PGF₂ (not shown). As observed for the C20 analogs, although the MS/MS spectra of the 13,14-dihydro-15-keto-2,3-dinor-5,6-dihydro metabolite and its 2,3-dinor-5,6-dihydro analog have the same mass-to-charge ratio for the precursor (m/z 327) and some product ions, the former can be clearly distinguished by the prominent ion at m/z 113, typical of all metabolites bearing the 13,14-dihydro-15-keto modification, and the absence of an ion at m/z 111 (Fig 6).

13,14-DIHYDRO-15-KETO-2,3,4,5-TETRANOR METABOLITE. A prominent product with this structure was found for PGF₂, and among F₂-iPs, for 8-epi-PGF₂, 15S-ent-8-epi-PGF₂, 12-epi-PGF₂, and ent-12-epi-PGF₂ (Table 1). For 15S-ent-8-epi-PGF₂, this was by far the most abundant metabolite. These compounds were identified by comparing their MS/MS spectra (Fig 5) with two authentic C20 and C18 homologs, 13,14-dihydro-15-keto-PGF₂ and 13,14-dihydro-15-keto-2,3-dinor-5,6-dihydro-ent-8-epi-PGF₂ (Fig 4 and Fig 6). As expected, the 13,14-dihydro-15-keto-2,3,4, 5-tetranor metabolite had the carboxylate anion (m/z 299) and most of the product ions at the same m/z value as the 2,3,4,5-tetranor metabolite, except for the expected major ion at m/z 113 (Fig 5). HPLC and GC behavior, as well as the NICIMS features of all isomers, were consistent with this putative structure (Table 2 and Table 3). The presence of the keto group was also confirmed, e.g., for 13,14-dihydro-15-keto-2,3,4,5-tetranor-8-epi-PGF₂, by observing that the two peaks (syn/anti isomers) eluting after 9.5 and 9.6 min at m/z 472 disappeared when the keto group was not derivatized to methoxime, whereas a peak appeared at m/z 443 after 9.7 min, as expected (not shown).

Taurine conjugates Taurine conjugates of parent PGF₂, F₂-iPs, and their prominent C20 and C18 metabolites were identified on the basis of their HPLC behavior ( Table 5 and Fig 8) and MS/MS spectra ( Table 6, Fig 8, and Fig 9). Some of these spectra showed striking similarities with those described by Hankin et al. (20) for the corresponding taurine conjugates of PGE₂ and its metabolites. Because no reference compounds were available, we were not able to quantify these metabolites. However, taking the intensity of the total ion current generated by each of these compounds on HPLC-MS/MS as an approximate indicator of its relative abundance, for each diastereoisomer the total amount of taurine conjugates and unconjugated products was of about the same order of magnitude. This is in accordance with Hankin's study (20), showing comparable overall amounts of radioactivity associated with taurine-conjugated or unconjugated metabolites of ³H-PGE₂ formed by isolated rat hepatocytes.

View larger version (24K): [in this window] [in a new window]   Figure 8. HPLC-MS/MS tracing of TIC and MS/MS spectra obtained from collision-activated decomposition of the molecular anions (m/z 460; 56 eV) of hepatocyte-derived 1-tauro-PGF2, and 1-tauro-13,14-dihydro-15-keto-PGF2. HPLC retention time is indicated for each metabolite.

View larger version (24K): [in this window] [in a new window]   Figure 9. MS/MS spectra obtained from collision-activated decomposition of the molecular anions of hepatocyte-derived 1-tauro-2,3-dinor-8-epi-PGF2 and 1-tauro-13,14-dihydro-15-keto-2,3-dinor-8-epi-PGF2 (m/z 432; 42 eV) plus 1-tauro-2,3-dinor-5,6-dihydro-PGF2 and 1-tauro-13,14-dihydro-15-keto-2,3-dinor-5,6-dihydro-PGF2 (m/z 434; 56 eV). HPLC retention time is indicated for each metabolite.

View this table: [in this window] [in a new window]   Table 5. HPLC-MS/MS behavior of taurine conjugates of PGF2, Class III F2-iPs and their metabolites

View this table: [in this window] [in a new window]   Table 6. Product ions detected by MS/MS after fragmentation of molecular anions [M-H]- of taurine conjugates of PGF2, Class III F2-iPs and their metabolites

Excluding the taurine conjugates of 2,3-dinor and 13,14-dihydro-15-keto-2,3-dinor metabolites which gave a peculiar fragmentation, like their unconjugated counterparts, the MS/MS spectra of taurine conjugates showed: 1) prominent ions derived from detachment of the entire or fragmented taurine group (m/z 124, H₂N-CH₂-CH₂-SO₃^- m/z 107, CH₂ = CH-SO₃^- m/z 80, SO₃^-< o 2) ions derived from retention of the charge on the PGF backbone with loss of H₂O and/or C₂H₄O (Table 6). Other prominent ions correspond to fragments of the -chain conjugated with taurine (for the parent compounds, m/z 151, 178, and 233, corresponding to taurine attached to C1, C1–C3, and C1–C7). Of these, the smallest fragment at m/z 151 (CONH(CH₂)₂SO₃^- oµµo o +° -'' o> A o ß H > -'' o o o E₂ and its metabolites, these products tend to be scarcely fragmented under standard MS/MS conditions, except for the taurine-dinor metabolite, which shows easy detachment of the taurine group, but no other significant fragment except at m/z 124. High collision energy (56 eV) was therefore used to obtain structurally informative MS/MS spectra of the taurine conjugates, except for 1-tauro-2,3-dinor and 1-tauro-13,14-dihydro-15-keto-2,3-dinor metabolites (42 eV).

As observed for unconjugated metabolites, the 13,14-dihydro-15-keto modification does not alter the molecular weight, so that MS/MS spectra have the molecular anions and many fragment ions at the same m/z values. However, a common feature of the MS/MS spectra of all tauro-conjugates with the 13,14-dihydro-15-keto modification was the absence of prominent ions in the central part of the mass spectrum (Table 6, and Fig 8 and Fig 9).

Taurine conjugates of c20 compounds. A taurine conjugate of the parent compound was observed for PGF₂ and all F₂-iP diastereoisomers (Table 1). This was by far the most abundant of all conjugates for all diastereoisomers, except for 15S-ent-8-epi-PGF₂, which, instead, had a more prominent 1-tauro-13,14-dihydro-15-keto metabolite, in accordance with 15-OH dehydrogenation being a preferential pathway for this diastereoisomer. The least abundant conjugate was found for 15S-ent-12-epi-PGF₂, in agreement with its scarce overall metabolism. In addition to the prominent 1-tauro-13,14-dihydro-15-keto metabolite of 15S-ent-8-epi-PGF₂, other 1-tauro-13,14-dihydro-15-keto-metabolites were found as minor products of the isomers forming the corresponding unconjugated metabolite, i.e., PGF₂ and 8-epi-PGF₂, (Table 1). This was taken in addition to the chromatographic and mass spectral behavior (Table 5 and Table 6, and Fig 8), as further proof of their identity in the absence of reference material.

Taurine conjugates of c18 metabolites. Taurine conjugates were identified for all major diastereoisomeric C18 metabolites (Table 1). In particular, we identified: 1) 1-tauro-2,3-dinor metabolites of 8-epi-PGF₂, 15R-8-epi-PGF₂, 12-epi-PGF₂, and ent-12-epi-PGF₂; 2) 1-tauro-2,3-dinor-5,6-dihydro metabolites of PGF₂, 12-epi-PGF₂ and ent-12-epi-PGF₂; 3) 1-tauro-13,14-dihydro-15-keto-2,3-dinor metabolites of 8-epi-PGF₂ (minor metabolite); and 4) 1-tauro-13,14-dihydro-15-keto-2,3-dinor-5,6-dihydro metabolites of PGF₂. HPLC-MS/MS features of these metabolites are shown in Table 5, and representative MS/MS spectra are shown in Fig 9 and interpreted in Table 6.

Taurine conjugates of c16 metabolites. Although MS/MS spectra of sufficient intensity could not be recorded for taurine conjugates of C16 metabolites, preliminary evidence obtained by multiple reaction monitoring of the precursor-to-product ion transition from m/z 406 to m/z 124 suggests that very small amounts of these conjugates were present in the hepatocyte medium. The most prominent unconjugated C16 metabolites with and without the 13,14-dihydro-15-keto modification had their corresponding taurine conjugate as a small product (data not shown). Hankin et al. (20) also found that the taurine conjugate of tetranor-PGE₂ was a minor component of the metabolic profile.

Metabolic profiles of PGF₂ and F₂-iPs
In a first experiment to establish the best experimental conditions for the identification study, PGF₂ and 8-epi-PGF₂ were incubated separately for 10, 20, and 40 min. Quantitation by GC-NICIMS of unconjugated metabolites in the incubation medium and cell lysate indicated that: 1) all unconjugated metabolites accumulated with time in the medium (sum of metabolites formed per plate: 5–10 ng/min), while their abundance remained constant in time in the cell lysate; and 2) the amount of the parent compounds decreased with time in the medium, reaching 150 ng/ml at 40 min, but was low at any time in the hepatocyte lysate (<20 ng).

Other GC-NICIMS measurements showed that, after 20 min of incubation, the unmetabolized compound in the medium was reduced to 50–60% of the initial amount for PGF₂, 8-epi-PGF₂, and 12-epi-PGF₂, 70% for 15R-8-epi-PGF₂, ent-12-epi-PGF₂, and 15R-12-epi-PGF₂, 80% for ent-8-epi-PGF₂ and 15S-ent-8-epi-PGF₂, and 95% for 15S-ent-12-epi-PGF₂. This is in agreement with the abundance profile of total metabolites (unconjugated plus conjugated) from the various diastereoisomers (PGF₂ 8-epi-PGF₂ 12-epi-PGF₂ >15R-8-epi-PGF₂ ent-12-epi-PGF₂ 15R-12-epi-PGF₂ ent-8-epi-PGF₂ 15S-ent-8-epi- PGF₂ >> 15S-ent-12-epi- PGF₂).

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

F₂-isoprostanes, a family of 64 compounds isomeric to PGF₂, are products of free radical-mediated non-enzymatic peroxidation of arachidonic acid (1) (2) (3). These compounds can be used to evaluate local or systemic lipid peroxidation in vivo, since they are detected esterified to tissue and plasma lipids and in the free form in body fluids (2) (3) (4) (5). Increased F₂-iP levels were found in several experimental and clinical conditions associated with free radical-mediated oxidant damage (2) (3) (4) (5). Given that F₂-iPs are becoming increasingly popular as oxidative stress markers, it is important to expand our knowledge about their formation and fate in vivo. Identification of the major metabolites of the various F₂-iP isomers, with the development of specific assays, may help evaluate the in vivo formation of selected compounds. This is of particular interest for the Type III regioisomer family, since the only compounds with proven biological activity so far are these diastereoisomers of PGF₂.

Isolated hepatocytes represent a well-characterized, rather simple system to screen for potential pathways of hepatic metabolism of endogenous and exogenous compounds (23). This in vitro system has the advantage of employing freshly isolated cells, with their intact enzymatic machinery, that reproducibly yield enough material for mass spectrometry identification of metabolites after rather simple purification procedures. This technique has been successfully used in the field of eicosanoids and isoprostanes (9) (17) (20).

We compared the metabolic fate of PGF₂ versus Type III F₂-iPs. Among the 16 possible diastereoisomers of the Type III family, we considered only the eight cis ring-substituted diastereoisomers that are believed to be the most abundant (6). We identified several products resulting from a combination of ß-oxidation, reduction of ⁵ and/or ¹³ double bonds, or 15-OH oxidation, plus taurine conjugates of parent compounds and their prominent C20 and C18 metabolites.

This study showed slight-to-marked stereo- and enantio-selectivity in the different enzymatic steps involved in the metabolic transformations of PGF₂ and Type III F₂-iPs. Some compounds were extensively metabolized to a variety of products, while others were poorly metabolized, with specific stereochemical configurations favoring or impeding selected reactions. For example, the steric configuration of the hydroxyl at C-15 must be S, as in the cyclooxygenase-derived PG, for 15-OH dehydrogenation to occur easily. In fact, no metabolites with the 15-keto modification (unconjugated or conjugated) were formed from diastereoisomers with the 15R configuration, except for the 13,14-dihydro-15-keto-2,3,4,5-tetranor metabolites of ent-8-epi-PGF₂ (minor product) and ent-12-epi-PGF₂. The ¹³ double bond of prostaglandin (PG) cannot be reduced without prior 15-OH dehydrogenation (17). This appears to be true for F₂-iPs too, as all metabolites with the 13,14-dihydro modification had the concomitant presence of the 15-keto group.

Another reaction that was markedly influenced by the steric structure was the reduction of ⁵ double bond, which proceeded easily for PGF₂, but was more or less impeded for the various enantiomeric pairs of F₂-iPs. The pathways that were the least affected by the stereochemical configuration were ß-oxidation and taurine conjugation, which gave rise to at least one product each, even for the scantly metabolized 15S-ent-12-epi-PGF₂.

The finding of significant amounts of taurine conjugates of PGF₂, F₂-iPs, and their prominent C20 and C18 metabolites drew our attention to the fact that, apart from the 13,14-dihydro-15-keto metabolites, all the other products identified here are formed through activation of the carboxyl group to a CoA-thioester. It is generally accepted that both ß-oxidation and taurine conjugation of carboxylic acids occur after the formation of a reactive CoA thioester intermediate (24). For PGF₂ and F₂-iPs, this common precursor appears to be easily formed for C20 and C18 products, because there is subsequent formation of 1) unconjugated C18 and C16 products through ß-oxidation, and 2) taurine conjugates of C20 and C18 products through taurine-N-acyltranferase activity. The fact that ß-oxidation does not proceed further to C14 products and that the C16 metabolites, although abundant, are poorly conjugated with taurine, suggests, among other possibilities, a scant formation of the precursor CoA-thioester of the C16 metabolites. Due to the presence of this common activation step for most of the prominent metabolites of PGF₂ and F₂-iPs, xenobiotic carboxylic acids that are metabolized through the corresponding CoA-thioesters might cause inhibition and/or diversion of PGF₂ and F₂-iPs metabolism in vivo. An example of such drugs are the widely used 2-arylpropionic non-steroidal anti-inflammatory drugs (24). This should be taken into consideration when measurements of F2-iPs or their selected metabolites in body fluids are used for assessing their formation in vivo.

Although a detailed quantitative description of PGF₂ and F₂-iP metabolism by isolated rat hepatocytes was beyond the scope of this study, for each diastereoisomer the total amounts of conjugated and unconjugated products were in general of the same order of magnitude. Although this cannot be taken as proof that these products are of comparable abundance in vivo, it certainly shows that these cells have an efficient enzymatic machinery for both pathways, and that these metabolites may therefore be formed in vivo, at least in the rat.

Many in vivo studies in different mammalian species found that exogenous PGF₂ is metabolized through common enzymatic pathways, including those discussed above (14) (15) (16) (18) (19). Nevertheless, not only are the prominent circulating and urinary metabolites different in the various species, but even in the same species the mode of administration may alter the metabolic profile of PGF₂ (16) (18) (19). Searching for the major metabolites of the endogenously formed, rather than exogenously administered, compounds in each species would hence be the best approach, but this can be extremely difficult in the absence of any preliminary analytical characterization of potential in vivo metabolites. A simplified approach such as that described here, despite suffering from the obvious intrinsic limitations of an in vitro study, allows, in principle, the obtaining of useful information for subsequent in vivo studies aimed at identifying the main endogenous products.

If their presence is proved in vivo, some of the metabolites identified here might become useful targets of new assays for determining overall F2-iP formation, or for evaluating the in vivo formation and fate of a specific bioactive diastereoisomer. Selected metabolites might also be chosen for their particular steric configurations or their greater abundance in vivo, for developing immunoassays with minimal cross-reactivity against endogenous cyclooxygenase-derived metabolites.

In conclusion, we showed how the different Type III F₂-iP diastereoisomers are degraded through pathways of PGF₂ metabolism, with metabolic profiles depending on their steric configuration. These data, together with our structural and analytical characterization of the metabolites, may be useful as a basis for further investigating F₂-iP disposition in vivo.

	FOOTNOTES

² Also affiliated with CNR, Cellular and Molecular Pharmacology Center, Via Vanvitelli, 32, 20129 Milano, Italy.

	ACKNOWLEDGMENTS

Claudia Rivalta is a fellow of the Fondazione A. e A. Valenti.

Manuscript received October 2, 2001; and in revised form November 19, 2001

Abbreviations: GC, gas chromatography; iP, isoprostane; MO, methoxime; MS, mass spectrometry; MS/MS, tandem mass spectrometry; NICI, negative ion chemical ionization; PFB, pentafluorobenzyl ester; PG, prostaglandin; SIR, selected ion recording; SPE, solid phase extraction; TMS, trimethylsilyl ether

	REFERENCES

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

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