Mechanisms for the Formation of Isoprostane Endoperoxides from Arachidonic Acid

"DIOXETANE" INTERMEDIATE VERSUS beta -FRAGMENTATION OF PEROXYL RADICALS*

Huiyong YinDagger §, Christine M. HavrillaDagger , Ling Gao, Jason D. Morrow||, and Ned A. PorterDagger **

From the Departments of Dagger  Chemistry and  Pharmacology, Center in Molecular Toxicology, Vanderbilt University, Nashville, Tennessee 37235

Received for publication, January 20, 2003, and in revised form, February 21, 2003

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The isoprostanes are a class of autoxidation products generated from arachidonic acid (or its esters) by a free radical initiated process. The potent biological activity of these compounds has been attracting intense research interest since they were detected in humans as well as animal models in the early 1990s. The measurement of these compounds has been regarded as one of the most useful non-invasive biomarkers for oxidative stress status. Two mechanisms for the formation of these compounds have been proposed. In the first mechanism, a peroxyl radical undergoes successive 5-exo cyclizations analogous to the enzymatic mechanism proposed for prostaglandin biosynthesis. The second mechanism starts with a 4-exo cyclization of a peroxyl radical leading to an intermediate dioxetane, a mechanism that has also been proposed for prostaglandin biosynthesis as well as for the formation of 4-hydroxy nonenal (HNE). Autoxidation of cholesteryl-15-HpETE under free radical conditions provides Type IV isoprostanes. The "dioxetane" mechanism for isoprostane generation from 15-HpETE requires that optically pure products are formed from an optically pure reactant, whereas an alternate mechanism for the process involving beta -fragmentation of the 15-peroxyl would give racemic isoprostane products. We have carried out a test of the mechanism based upon these stereochemical requirements. The results of analysis of the product mixture derived from autoxidation of optically pure Ch-15-HpETE by atmospheric pressure chemical ionization-mass spectrometry coupled with chiral high performance liquid chromatography indicate that the major isoprostane diastereomers are formed as a racemic mixture. These experimental results are consistent with a mechanism for isoprostane formation involving beta -fragmentation of the 15-peroxyl radical followed by re-addition of oxygen to form the 11-HPETE peroxyl, and they exclude a mechanism proceeding through the formation of a dioxetane intermediate.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The oxidation products derived from arachidonic acid play an important role in the control of organ physiology (1-3). The bicyclic endoperoxide prostaglandin G2 (PGG2)1 is formed from the oxidation of arachidonic acid by cyclooxygenases (COX enzymes). This endoperoxide and its reduced form at C-15, PGH2, can serve as a precursor to a variety of biologically active compounds, such as thromboxane (TXA2) (4), the prostaglandins PGF2, PGD2, and PGE2, and prostacyclin (PGI2) (5, 6). These compounds have a broad range of biological function and activity (1, 5).

Free radical oxidation of arachidonic acid or arachidonate esters in vitro and in vivo (7, 8) gives a series of compounds (isoprostanes) that are stereoisomers of PGF2alpha after reduction of the bicyclic endoperoxides (9). In theory, four types of bicyclic endoperoxides can be generated, depending upon the initial radical abstraction of the possible bisallylic hydrogens at C-7, C-10, or C-13 of the arachidonate (Scheme 1). Isoprostanes are formed from the reduction of intermediate bicyclic endoperoxides enzymatically or by chemical means. A total of 64 stereoisomers of isoprostanes can be generated from the oxidation of arachidonate. The measurement of these isoprostanes has been employed as a reliable index for oxidative stress (10-12).


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Scheme 1.  

The enzymatic oxidation of arachidonic acid by cyclooxygenase enzymes generates optical pure bicyclic endoperoxides, whereas the uncontrolled free radical oxidation process leading to the isoprostanes produces racemic mixtures. Furthermore, a variety of other highly oxidized products, such as monocyclic peroxides, serial cyclic peroxides, and a novel compound having a bicyclic endoperoxide moiety and a cyclic peroxide structure are detected from the non-enzymatic reactions (9, 13, 14).

The oxidation products of pure hydroperoxides of cholesteryl arachidonate have also been characterized by MS techniques. For example, the 11-HpETE hydroperoxide leads specifically to Type IV isoprostanes, and the stereochemistry of the type IV bicyclic endoperoxides generated from the cholesteryl ester 11-HpETE (Ch-11-HpETE) was extensively studied (9). Eight possible diastereoisomers were detected by the use of a GC-MS technique by comparison of products with known synthetic standards. Ch-15-HpETE also gives rise to Type IV isoprostanes, and a free radical mechanism based on beta -fragmentation of the 15-peroxyl radical was proposed to explain the experimental facts (Scheme 2a) (14, 15). The key step in these transformations lies in the beta -fragmentation of the 15-peroxyl radical to generate a pentadienyl radical followed by an O2 re-addition at C-11 to give rise to the 11-peroxyl radical, which then undergoes subsequent cyclization to generate the bicyclic endoperoxides.


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Scheme 2.  

A mechanism for prostaglandin biosynthesis starting from a 15-HpETE peroxyl radical was proposed by Corey and Wang (16) based on the formation of a "dioxetane" intermediate. In this mechanism, the 15-peroxyl radical undergoes 4-exo cyclization to give a dioxetane intermediate as shown in Scheme 2b. This mechanism has been proposed by Rokach et al. (2) and others (17-21) as an alternative to explain the formation of regioisomeric bicyclic endoperoxides from an arachidonyl-containing lipid.

The conversion of qinghao (artemisinic) acid, an antimalarial compound extracted from a Chinese plant, to dehydroqinghaosu (artemisitene) was initially proposed to occur via the 4-exo cyclization of an allylic peroxyl radical to form a dioxetane intermediate (22-24). Later studies suggested that this transformation was better explained by oxidation of an intermediate enol under the reaction of copper (II) trifluoromethanesulfonate (25).

The formation of 4-hydroxy-2E-nonenal (4-HNE) from hydroperoxides of linoleic acid was first proposed to occur via a dioxetane intermediate (26, 27). Several alternative mechanisms for the formation of HNE, a reactive molecule that has a possible role in several human diseases, have been invoked (28-31). But little experimental evidence has been presented that either supports or denies the dioxetane mechanism of HNE formation (32, 33).

We have carried out a set of experiments designed to test the two mechanistic proposals presented in Schemes 2, a and b. If optically pure starting material, Ch-15-HpETE, is subjected to autoxidation, beta -fragmentation of the intermediate 15-peroxyl radical and re-addition of oxygen gives a racemic C-11 peroxyl radical that will lead to a mixture of isoprostane diastereomers, all of which will be racemic. If the dioxetane mechanism operates, a mixture of optically pure isoprostane diastereomers is anticipated. Our results favor the beta -fragmentation mechanism shown in Scheme 2a.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Materials-- All lipid autoxidation reactions were carried out under an atmosphere of oxygen unless otherwise noted. Air and argon were passed through a bed of calcium sulfate desiccant. Benzene was distilled from sodium and stored over 4-A molecular sieves. Tetrahydrofuran and dichloromethane were dried by Solv-Tek (Berryville, VA) solvent purification columns using activated alumina for drying and Q-5 packing for deoxygenating the solvents.

Oxygen (medical grade) was obtained from A.L. Compressed Gas (Nashville, TN). HPLC-grade solvents were purchased from Burdick & Jackson (Muskegon, MI) or EM Science (Gibbstown, NJ). All lipids were purchased from Nu Chek Prep (Elysian, MI) and were of the highest purity (>99+%). Soybean lipoxygenase (Type I-B, EC. 1.13.11.12) and SigmaUltra-grade boric acid were purchased from Sigma. The free radical initiator di-tert-butyl hyponitrite (DTBN) was synthesized prior to use (34, 35). N,O-Bis-(trimethylsilyl)trifluoroacetamide (BSTFA) was purchased from Superlco Inc. (Bellefonte, PA). PGF2alpha and PGF2alpha -d4 standards were purchased from Cayman Chemicals (Ann Arbor, MI). All other reagents were purchased from Aldrich and used without further purification.

Methods-- Reactions involving hydroperoxides were visualized by TLC using a stain of 1.5 g of N,N'-dimethyl-p-phenylenediamine dihydrochloride, 25 ml H2O/125 ml MeOH, and 1 ml acetic acid. Hydroperoxides yield an immediate pink color, whereas protected hydroperoxides exhibit a pink color after mild charring. General TLC staining was accomplished by iodine or the use of a phosphomolybdic acid stain prepared as a 20% (w/v) solution in EtOH. In general, hydroperoxides were stored as dilute solutions with one mole percent butylated hydroxytoluene (BHT) in either hexanes or benzene at -78 °C and were never exposed to temperatures >40 °C.

Flash column chromatography was performed using 35-70 µm of silica gel. Thin layer chromatography was performed using a 0.2-mm thick layer of silica gel-coated aluminum (60 F254, EM Industries), and TLC plates were analyzed using UV light (254 nm) with a Mineralight UVSL-25 hand lamp. Preparative TLC was performed on silica gel 60ALK6D plates (Whatman).

Analytical HPLC was carried out using a Waters Model 600E pump with a Waters 996 Photodiode array detector. Millenium32 chromatography software (Waters Corp., Milford, MA) was used to control the 996 detector and collect and process data. Cyclic peroxide analysis by analytical HPLC utilized a single Beckman Ultrasphere 5-µm (4.6 mm × 25 cm) silica column. A flow rate of 1 ml/min was used for analytical normal phase HPLC. Preparative HPLC was performed using a Dynamax-60 Å 8 µm (83-121-C) silica column (21.4 mm × 25 cm) with a flow rate of 10 ml/min. Narrowbore HPLC for MS analysis used a single Beckman Ultrasphere 5-µm (2.0 mm × 25 cm) silica column for analysis of cyclic peroxides and two Beckman Ultrasphere 5 µm (2.0 mm × 25 cm) silica columns for acyclic hydroperoxide analysis.

Mass Spectrometry-- Coordination ion spray mass spectrometry (CIS-MS) was accomplished using a Finnigan TSQ-7000 (San Jose, CA) triple quadrupole mass spectrometer operating in positive ion mode equipped with a standard API-1 electrospray ionization (ESI) source. The source was outfitted with a 100-µm deactivated fused silica capillary. Data acquisition and evaluation were conducted on ICIS EXECUTIVE INST, version 8.3.2, and TSQ 7000 software INST, version 8.3. Data collected for selected reaction monitoring (SRM) experiments was also processed using Xcalibur, version 1 (Finnigan, San Jose, CA).

Nitrogen gas served both as sheath gas and auxiliary gas; argon served as the collision gas. The electrospray needle was maintained at 4.6 kV, and the heated capillary temperature was 200 °C. The tube lens potential and capillary voltage were optimized to maximize ion current for electrospray, with the optimal determined to be 80V and 20V, respectively, for cholesteryl ester analysis. Positive ions were detected scanning from 100-1000 atomic mass units with a scan duration of 1 s. Profile data was recorded for 1 min and averaged for analysis. For collision-induced dissociation (CID) experiments, the collision gas pressure was set from 2.30 to 2.56 millitorr. To obtain fragmentation information of each compound, the dependence of offset voltage and relative ion current was studied. The collision energy offset was varied from 10 to 40 eV depending on the compound being analyzed.

Samples were introduced either by direct liquid infusion or by HPLC. For direct liquid injection, stock solutions of the lipids (100 ng/µl in 1% IPA in hexane) were prepared and mixed 1:1 with silver tetrafluoroborate (51.4 ng/µl in IPA). Samples were introduced to the ESI source by syringe pump at a rate of 10 µl/min. For HPLC sample introduction a Hewlett-Packard 1090 HPLC system was used. The auxiliary gas flow rate to the ESI interface was increased to between 5 and 10 units to assist in desolvation of the samples. For cholesteryl arachidonate hydroperoxide analysis, normal phase HPLC sample introduction was carried out using two tandem Beckman Ultrasphere narrowbore 5-µm silica columns (2.0 mm × 25 cm) operated in isocratic mode with 0.35% isopropanol in hexanes. For analysis of cyclic peroxide mixtures, sample introduction was carried out using a single Beckman Ultrasphere narrowbore 5-µm silica column (2.0 mm × 25 cm) operated in isocratic mode with 1.0% isopropanol in hexanes. The flow rate for both modes of chromatography was 150 µl/min. Column effluent was passed through an Applied Biosystems 785A Programmable Absorbance UV detector with detection at 234 nm. An Upchurch high pressure mixing tee was connected next in series for the post column addition of the silver salts. The silver tetrafluoroborate (AgBF4) solution (0.25 mM in isopropanol) was added via a Harvard Apparatus (Cambridge, MA) syringe pump at a flow rate of 75 µl/min. A long section of PEEK tubing (1.04 m, 0.25-mm internal diameter) allowed time for the complexation of the silver to the lipid while delivering effluent to the mass spectrometer. A Rheodyne 7725 injector was fitted with a 100-µl PEEK loop for 20-50-µl sample injections.

LC-APCI ECNI-MS was performed on the same instrument TSQ-7000 using an APCI ion source. The mass spectrometers operating conditions were as follows: vaporizer temperature, 475 °C; heated capillary, 300 °C; and the corona discharge needle was set at 16 µA (36). The pressure of sheath gas and auxiliary gas was optimized for maximal response. For full scan and SRM analyses, unit resolution was maintained for both parent and daughter ions.

GC-NICI MS was performed using a Hewlett-Packard HP5989A GC-MS instrument interfaced with an IBM Pentium II computer system. GC was performed using a 30 m, 0.25-mm diameter, 0.25-µm-film thickness, DB-WAX column (J & W Scientific, Folsom, CA) or 15 m, 0.25-mm diameter, 0.25-µm film thickness, DB 1701 fused silica capillary column (Fison, Folsom, CA). The column temperature was programmed from 190 to 260 °C at 10 °C/min. Methane was used as the carrier gas at a flow rate of 1 ml/min. Ion source temperature was 250 °C, electron energy was 70 eV, and filament current was 0.25 mA. Lipid autoxidations were performed at 37.0 ± 0.2 °C, and temperature was controlled using an I2R Thermo-watch ML6-1000SS.

Autoxidation of the Hydroperoxides of Cholesteryl Arachidonate-- Ch-15(S)-HpETE made by a chemo-enzymatic method was converted to more highly oxidized peroxides according to the literature (14). The product oxidation mixture was obtained by incubation of Ch-15-HpETE under free radical conditions using DTBN as an initiator. A typical oxidation experiment was carried out as follows. Approximately 10 mg of Ch-15-HpETE was mixed with 10% (molar) of DTBN in 1 ml of anhydrous benzene. The mixture was stirred at 37 °C under an air atmosphere for 24 h. The oxidized mixture was diluted in benzene and stored at -78 °C with butylated hydroxytoluene. An aliquot of the mixture was analyzed by normal phase HPLC using 1.0% IPA in hexanes.

Derivatization of the Oxidation Products of Cholesteryl Arachidonate for GC-MS Studies-- To 100 µl of the oxidation mixture was added 10 ng of PGF2alpha -d4 as internal standard. An excess amount of PPh3 or SnCl2 was added to the mixture. After evaporation of the solvent, the residue was dissolved in 200 µl of ethanol, and to the solution was added 200 µl of 8 M aqueous KOH solution. After stirring at 40 °C for an hour, the mixture was cooled to room temperature, and the pH of the solution was adjusted to 3 by adding dilute HCl. The aqueous solution was extracted by ethyl acetate three times. The combined organic phases were dried over Na2SO4. After evaporation of the solvent, the residue was dissolved in 20 µl of CH3CN. To the resulting solution was added 20 µl of 10% (v/v) pentafluorobenzyl bromide in acetonitrile and 10 µl of 10% (v/v) N,N-diisopropylethylamine in acetonitrile, and the mixture was kept at room temperature for 30 min. The reagent was dried under nitrogen, and the residue was subjected to TLC separation using the solvent ethyl acetate/methanol (98:2; v/v). Approximately 5 µg of the pentafluorobenzyl (PFB) ester of PGF2alpha was applied on a separate lane and visualized by spraying with a 10% solution of phosphomolybdic acid in ethanol followed by heating. Compounds migrating in the region of the PFB ester of PGF2alpha (Rf = 0.4) and the adjacent area 2 cm above and below were scraped and extracted from the silica gel by ethyl acetate. The PFB esters were also separated by normal phase HPLC using dual silica columns. The solvent used for this purpose is 12% IPA in hexanes. The chiral LC-APCI-MS experiments were conducted using a Chiralpak AD column (25 × 0.46 cm, Chiral Technologies, Exton, PA) and 8% IPA in hexanes.

After evaporation of the ethyl acetate, 20 µl of BSTFA and 10 µl of dimethylformamide were added to the residue, and the mixture was incubated at 40 °C for 20 min. The reagents were dried under nitrogen, and the derivatives were dissolved in 10 µl of dry undecane for analysis of GC-MS.

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The optically pure Ch-15(S)-HpETE was synthesized by a chemo-enzymatic route as described previously (14). The optical purity (enantiomeric excess) value of the hydroperoxide was determined by chiral HPLC method developed by Brash and co-workers (37). Thus, Ch-15(S)-HpETE used in these studies was reduced by PPh3 to the corresponding alcohol (Ch-15(S)-HETE), which was converted to the methyl ester by reaction with NaOMe. HPLC analysis of methyl-15(S)-HETE prepared in this way by the use of a Chiralpak AD column showed that the Ch-15(S)-HpETE had 98.9% enantiomeric excess (Fig. 1).


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Fig. 1.   Separation of 15-HpETE Me by chiral HPLC. a, racemic mixture of HETE Me. b, soybean lipoxygenase (SLO) product.

Ch-15(S)-HpETE was incubated for 24 h at 37 °C under oxygen using DTBN as a radical initiator. The product mixture resulting from this incubation was analyzed by LC-MS using the silver ion coordination MS technique (Ag+ CIS-MS) technique described previously (14). In addition to bicyclic endoperoxides, monocyclic and serial cyclic peroxides were also observed in this mixture. SRM results of such an analysis are illustrated in Fig. 2. The SRM experiments were carried out by selection of a specific parent ion, fragmentation of that ion, and detection of fragments characteristic of a particular structure. Panel a of Fig. 2 shows the UV absorption of the oxidation mixture at 234 nm, a wavelength characteristic of the conjugated diene chromophore. Panel b shows the SRM chromatogram for Ch-15-HPETE, the starting material, whereas detection of Ch-11-t,t-HpETE is shown in panel c.2 Formation of Ch-11-t,t-HpETE is consistent with beta -fragmentation of the 15-peroxyl radical and re-addition of oxygen at C-11 as shown in Scheme 2. An SRM chromatogram of bicyclic endoperoxides is shown in panel d, whereas monocyclic and serial cyclic peroxides are shown in panels e and f, respectively.3


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Fig. 2.   SRM results of oxidation products from Ch-15(S)-HpETE by LC-silver CIS/MS narrowbore silicon column, 1% IPA in hexane. m/z shown indicates silver adduct with isotope 107.

An aliquot of the crude reaction mixture from above was derivatized by reduction of PPh3 or SnCl2, followed by basic hydrolysis and conversion to the PFB ester. After conversion of any free OH groups to TMS derivatives, the mixture was analyzed by GC-ECNICI-MS using a DB-WAX GC column (38). This analysis, illustrated in Fig. 3, revealed that all eight possible isoprostane diastereoisomers were present in the reaction mixture.


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Fig. 3.   Selective ion monitoring (SIM; m/z 569) of isoprostanes derived from Ch-15(S)-HpETE studied by GC-ECNI-MS. GC, DB wax, 30 m, 190-260 °C, 10 °C/min.

The significant peaks shown in Fig. 3 were assigned by spiking the mixture with independently synthesized standards. The diastereoisomers with cis dioxolane side chains were formed preferentially over those with trans side chains (9). The ratio of PGF2alpha (peak 6) and 12-epi PGF2alpha (peak 8) is 1:3, which is consistent with the previous observations (16).

The crude reaction mixture derived from Ch-15(S)-HpETE was reduced by SnCl2, the extracted organic phase was subjected to basic hydrolysis, and the product acids were converted to their PFB esters. After separation by TLC with a solvent mixture of 98:2 (ethyl acetate/methanol), isoprostane PFB esters was further separated into isomerically pure fractions by normal phase HPLC using tandem analytical silica gel columns with 12% isopropanol in hexane. SnCl2 was used to reduce the bicyclic endoperoxides because the product of PPh3 reaction, triphenyl phosphine oxide, elutes in the middle of the diastereoisomers of isoprostanes under the HPLC conditions used. A portion of each purified isoprostane PFB ester was converted to its TMS derivative for GC-ECNICI-MS analysis, which confirmed the isomeric purity and identity of each. The remainder of each PFB isoprostane fraction (not converted to the TMS) was analyzed by LC-APCI-MS using a chiral HPLC column. APCI-MS was employed to analyze the PFB esters of the isoprostanes because of its excellent sensitivity and selectivity of this method (36). A summary of the analysis protocol for the Ch-15(S)-HpETE product mixture is presented in Scheme 3.


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Scheme 3.  

The PFB esters of PGF2alpha are analyzed in the negative mode on a triquadrupole mass spectrometer equipped with an APCI ionization source. The Q1 scan experiment is carried out by scanning the ions in the first quadrupole. CID is run by selecting a particular parent ion (for example, m/z 353) in the first quadrupole and fragmenting it in the second by collision with argon. The resulting ions are scanned in the third quadrupole. A Q1 scan and CID spectra of the parent ion m/z 353 of the PFB ester of PGF2alpha are shown in Fig. 4. The CID fragments are essentially the same as those obtained from PGF2alpha using negative ion ESI-MS, and the fragment of m/z 193 is unique for Type IV isoprostanes (39).


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Fig. 4.   Q1 scan and CID of PGF2alpha PFB ester by APCI-ECNI-MS.

The four major isoprostane PFB ester stereoisomers, isolated as shown in Scheme 3, were analyzed by chiral HPLC/MS. SRM experiments were carried out by monitoring the characteristic fragmentation pathways from m/z 353 to 193, 309, and 247, respectively. Monitoring of the three fragmentation reactions gave essentially the same chromatographic results. Only the m/z 353 to 193 SRM chromatogram is shown in Fig. 5.


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Fig. 5.   SRM results of fraction separated from autoxidation mixture of 15(S)-HpETE Ch by chiral LC-APCI-ECNI-MS. SRM, m/z 353 to 193. LC, 8% IPA in hexane, Chiral-Pak AD, 4.6 cm, 1 ml/min. Top, chromatogram, reaction mixture. Bottom, chromatograms, optically pure enantiomers. a, 8,15-di-epi PGF2alpha ; b, 12-epi PGF2alpha .

The SRM chromatograms in Fig. 5, a (8,15-di-epi PGF2alpha ) and b (12-epi PGF2alpha ), showed that both of these isoprostanes are formed as a racemic mixture. The absolute configuration of the enantiomers was assigned by comparison with independently synthesized standards, as shown in Fig. 5. The enantiomers of 8-epi PGF2alpha and 12,15-di-epi PGF2alpha , all of which are available by chemical synthesis, do not separate on a Chiralpak AD, OD, or OJ column.

    DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The results presented here provide evidence that suggests that the dioxetane mechanism for isoprostane formation does not operate. This mechanism, which is based upon a proposal for prostaglandin biosynthesis and is shown in Scheme 2b, has been suggested as one of two possible mechanisms for isoprostane formation. In the dioxetane mechanism, the stereogenic center at C-15 of the substrate is carried through the sequence unchanged and, consequently, the isoprostane formed should be optically pure given an optically pure starting material.

The beta -fragmentation mechanism shown in Scheme 2a requires, in contrast to the dioxetane mechanism, that a racemic mixture of isoprostanes is produced from the reaction of optically pure Ch-15(S)-HpETE. The stereogenic center is lost in the beta -fragmentation step of the C-15 peroxyl radical, and one anticipates equal amounts of both enantiomers upon addition of oxygen at C-11 and the formation of the new stereogenic center. The chiral LC-APCI-ECNI-MS results shown in Fig. 5 are thus entirely consistent with the beta -fragmentation mechanism. An optically pure hydroperoxide precursor gives rise to racemic isoprostane products (40).4

We note that the 4-exo peroxyl radical cyclization required in the dioxetane mechanism is energetically disfavored, and there is a decided lack of evidence in the literature to support such a reaction (26). Furthermore, the dioxetane mechanism does not account for the formation of other highly oxidized cyclic peroxides such as the monocyclic and serial cyclic peroxides shown in Fig. 2. The formation of these compounds is entirely consistent with the fragmentation reaction (Scheme 2) (13).

The two mechanisms predict different regioisomeric products starting from different HpETEs as shown in Scheme 4. Bicyclic endoperoxides from 11- or 9-HPETEs cannot be formed based on the dioxetane mechanism, whereas the beta -fragmentation mechanism predicts essentially the same isoprostane products starting from either the 15- or 11-HpETEs. This was previously confirmed in our experiments with regioisomeric HpETEs (13). For example, identical isoprostane products were observed starting from either Ch-15-HpETE or Ch-11-HpETE, although the distribution of those products is somewhat dependent on the starting compound (9).


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Scheme 4.  

The observation that racemic isoprostanes are formed from optically active HpETE precursors is of potential biological importance because it is predicted that different isoprostane enantiomers possess different biological activities (10-12). In addition, these findings suggest that optically pure HpETEs generated by the lipoxygenase family of enzymes can undergo cyclization resulting in the formation of racemic isoprostane products. This is likely relevant to the formation of isoprostanes in vivo, because deletion of the 12/15-lipoxygenase gene in genetically altered mice reduces isoprostane levels significantly (41).

In summary, two distinct mechanisms have been proposed to explain the formation of bicyclic endoperoxides from 15-HpETE. In the first mechanism, beta -fragmentation of the 15-peroxyl radical generates a pentadienyl radical. O2 addition to the pentadienyl at C-11 and subsequent 5-exo cyclization gives rise to the PGG2-like products. In the second mechanism, 4-exo cyclization of the 15-peroxyl radical generates a dioxetane intermediate, which proceeds on to isoprostane products. These two mechanisms have been extended to explain the formation of other regioisomeric endoperoxides from the free radical-induced oxidation of other arachidonate-containing lipids as well. The results of chiral analysis of the isoprostane products formed from an optically pure precursor, support the beta -fragmentation mechanism. This mechanism is also consistent with other observations, such as the formation of highly oxidized cyclic peroxides.

    ACKNOWLEDGEMENTS

We thank Dr. David Hachey, Ms. Lisa Mannier, and Mrs. Betty Fox of the Mass Spectrometry Research Center of Vanderbilt University for assistance with the MS analysis. We also thank Dr. Alan Brash for the chiral analysis of Me-15(S)-HETE.

    FOOTNOTES

* This work was supported by National Institutes of Health Grants HL17921, GM15431, and P30 ES00267 and National Science Foundation Grant CHE 9996188.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ Recipient of the Warren Graduate Fellowship of the Chemistry Department at Vanderbilt University.

|| Recipient of a Burroughs Wellcome Fund Clinical Scientist Award in Translational Research.

** To whom correspondence should be addressed: Dept. of Chemistry, Vanderbilt University, Box 1822, Station B, Nashville, TN 37235. Tel.: 615-343-2693; Fax: 615-343-5478; E-mail: n.porter@vanderbilt.edu.

Published, JBC Papers in Press, February 27, 2003, DOI 10.1074/jbc.M300604200

2 The fragments detected are the result of Hock fragmentation as described in Ref. 13; the assignment of structure to Ch-11-t, t-HPETE is tentative.

3 The fragments detected are the result of Hock fragmentation as described in Ref. 13.

4 We note that substantial racemization of starting hydroperoxides recovered from similar reactions was observed by O'Connor et al. (40), consistent with peroxyl radical-fragmentation.

    ABBREVIATIONS

The abbreviations used are: PGG2, prostaglandin G2 (also PGH2, PGF2, etc.); APCI, atmospheric pressure chemical ionization; BSTFA, N,O-bis-(trimethylsilyl)trifluoroacetamide; Ch-HpETE, cholesteryl hydroperoxyeicosatetraenoate; CID, collision induced dissociation; CIS, coordination ion spray; DTBN, di-tert-butyl hyponitrite; ECNI, electron capture negative ion; ECNICI, ECNI chemical ionization; ESI, electrospray ionization; GC, gas chromatography; HNE, 4-hydroxy nonenal; HPLC, high-performance liquid chromatography; IPA, isopropanol; LC, liquid chromatography; MS, mass spectrometry; PFB, pentafluorobenzyl; SRM, selective reaction monitoring; TLC, thin layer chromatography; TMS, trimethylsilyl.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

1. Rocca, B., and FitzGerald, G. A. (2002) Int. Immunopharmacol. 2, 603-630[CrossRef][Medline] [Order article via Infotrieve]
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