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• W2114305841 abstract "Free radical-initiated oxidant injury and lipid peroxidation have been implicated in a number of neural disorders. Docosahexaenoic acid is the most abundant unsaturated fatty acid in the central nervous system. We have shown previously that this 22-carbon fatty acid can yield, upon oxidation, isoprostane-like compounds termed neuroprostanes, with E/D-type prostane rings (E4/D4-neuroprostanes). Eicosanoids with E/D-type prostane rings are unstable and dehydrate to cyclopentenone-containing compounds possessing A-type and J-type prostane rings, respectively. We thus explored whether cyclopentenone neuroprostanes (A4/J4-neuroprostanes) are formed from the dehydration of E4/D4-neuroprostanes. Indeed, oxidation of docosahexaenoic acid in vitro increased levels of putative A4/J4-neuroprostanes 64-fold from 88 ± 43 to 5463 ± 2579 ng/mg docosahexaenoic acid. Chemical approaches and liquid chromatography/electrospray ionization tandem mass spectrometry definitively identified them as A4/J4-neuroprostanes. We subsequently showed these compounds are formed in significant amounts from a biological source, rat brain synaptosomes. A4/J4-neuroprostanes increased 13-fold, from a basal level of 89 ± 72 ng/mg protein to 1187 ± 217 ng/mg (n = 4), upon oxidation. We also detected these compounds in very large amounts in fresh brain tissue from rats at levels of 97 ± 25 ng/g brain tissue (n = 3) and from humans at levels of 98 ± 26 ng/g brain tissue (n = 5), quantities that are nearly an order of magnitude higher than other classes of neuroprostanes. Because of the fact that A4/J4-neuroprostanes contain highly reactive cyclopentenone ring structures, it would be predicted that they readily undergo Michael addition with glutathione and adduct covalently to proteins. Indeed, incubation of A4/J4-neuroprostanes in vitro with excess glutathione resulted in the formation of large amounts of adducts. Thus, these studies have identified novel, highly reactive A/J-ring isoprostane-like compounds that are derived from docosahexaenoic acid in vivo. Free radical-initiated oxidant injury and lipid peroxidation have been implicated in a number of neural disorders. Docosahexaenoic acid is the most abundant unsaturated fatty acid in the central nervous system. We have shown previously that this 22-carbon fatty acid can yield, upon oxidation, isoprostane-like compounds termed neuroprostanes, with E/D-type prostane rings (E4/D4-neuroprostanes). Eicosanoids with E/D-type prostane rings are unstable and dehydrate to cyclopentenone-containing compounds possessing A-type and J-type prostane rings, respectively. We thus explored whether cyclopentenone neuroprostanes (A4/J4-neuroprostanes) are formed from the dehydration of E4/D4-neuroprostanes. Indeed, oxidation of docosahexaenoic acid in vitro increased levels of putative A4/J4-neuroprostanes 64-fold from 88 ± 43 to 5463 ± 2579 ng/mg docosahexaenoic acid. Chemical approaches and liquid chromatography/electrospray ionization tandem mass spectrometry definitively identified them as A4/J4-neuroprostanes. We subsequently showed these compounds are formed in significant amounts from a biological source, rat brain synaptosomes. A4/J4-neuroprostanes increased 13-fold, from a basal level of 89 ± 72 ng/mg protein to 1187 ± 217 ng/mg (n = 4), upon oxidation. We also detected these compounds in very large amounts in fresh brain tissue from rats at levels of 97 ± 25 ng/g brain tissue (n = 3) and from humans at levels of 98 ± 26 ng/g brain tissue (n = 5), quantities that are nearly an order of magnitude higher than other classes of neuroprostanes. Because of the fact that A4/J4-neuroprostanes contain highly reactive cyclopentenone ring structures, it would be predicted that they readily undergo Michael addition with glutathione and adduct covalently to proteins. Indeed, incubation of A4/J4-neuroprostanes in vitro with excess glutathione resulted in the formation of large amounts of adducts. Thus, these studies have identified novel, highly reactive A/J-ring isoprostane-like compounds that are derived from docosahexaenoic acid in vivo. docosahexaenoic acid isoprostane neuroprostane prostaglandin 2,2′-azobis(2-amidinopropane) hydrochloride pentafluorobenzyl gas chromatography negative ion chemical ionization mass spectrometry high performance liquid chromatography liquid chromatography electrospray ionization collision-induced dissociation trimethylsilyl methylt-butyl ether N,O-bis(trimethylsilyl)trifluoroacetamide Docosahexaenoic acid (C22:6ω3, DHA)1 is estimated to account for ∼30% of the total fatty acids in brain tissue aminophospholipids (1Skinner E.R. Watt C. Besson J.A.O. Best P.V. Brain. 1993; 116: 717-725Crossref PubMed Scopus (117) Google Scholar, 2Carlson S.E. Semin. Neonatol. 2001; 5: 437-449Abstract Full Text PDF Scopus (107) Google Scholar). DHA is believed to be important for brain development (3Reich E.E. Zackert W.E. Brame C.J. Chen Y. Roberts II, L.J. Hachey D.L. Montine T.J. Morrow J.D. Biochemistry. 2000; 39: 2376-2383Crossref PubMed Scopus (68) Google Scholar), and its deficiency is associated with abnormalities in brain function (4Conner W.E. Neuringer M. Reisbick S. Nutr. Rev. 1992; 50: 21-29Crossref Scopus (211) Google Scholar). Oxidation of DHA has been an area of intense research because lipid peroxidation has been implicated in the pathogenesis of various central nervous system diseases, among them neurodegenerative disorders including Alzheimer's disease (3Reich E.E. Zackert W.E. Brame C.J. Chen Y. Roberts II, L.J. Hachey D.L. Montine T.J. Morrow J.D. Biochemistry. 2000; 39: 2376-2383Crossref PubMed Scopus (68) Google Scholar,5Halliwell B. Gutteridge J.M.C. Methods Enzymol. 1990; 186: 1-85Crossref PubMed Scopus (4428) Google Scholar, 6Markesbery W.R. Free Radic. Biol. Med. 1997; 23: 134-147Crossref PubMed Scopus (1966) Google Scholar, 7Knight J.A. Ann. Clin. Lab. Sci. 1997; 27: 11-25PubMed Google Scholar, 8Simonian N.A. Doyle J.T. Prog. Lipid Res. 1997; 36: 1-21Crossref PubMed Scopus (511) Google Scholar, 9Montine T.J. Markesbery W.R. Zackert W.E. Sanchez S.C. Roberts II, L.J. Morrow J.D. Am. J. Pathol. 1999; 155: 863-868Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar). We have reported previously (3Reich E.E. Zackert W.E. Brame C.J. Chen Y. Roberts II, L.J. Hachey D.L. Montine T.J. Morrow J.D. Biochemistry. 2000; 39: 2376-2383Crossref PubMed Scopus (68) Google Scholar, 10Roberts II, L.J. Montine T.J. Markesbery W.R. Tapper A.R. Hardy P. Chemtob S. Dettbarn W.D. Morrow J.D. J. Biol. Chem. 1998; 273: 13605-13612Abstract Full Text Full Text PDF PubMed Scopus (373) Google Scholar) that DHA is readily oxidized to generate 22-carbon isoprostane (IsoP)-like compounds, termed neuroprostanes (NPs). The formation of NPs from DHA is similar to the formation of IsoPs from arachidonic acid and proceeds via the generation of highly unstable endoperoxide intermediates (3Reich E.E. Zackert W.E. Brame C.J. Chen Y. Roberts II, L.J. Hachey D.L. Montine T.J. Morrow J.D. Biochemistry. 2000; 39: 2376-2383Crossref PubMed Scopus (68) Google Scholar, 10Roberts II, L.J. Montine T.J. Markesbery W.R. Tapper A.R. Hardy P. Chemtob S. Dettbarn W.D. Morrow J.D. J. Biol. Chem. 1998; 273: 13605-13612Abstract Full Text Full Text PDF PubMed Scopus (373) Google Scholar). These intermediates can undergo reduction in vitro and in vivo to form NPs containing F-type prostane rings (F4-NPs) or can isomerize to molecules with E-type and D-type prostane rings (E4/D4-NPs). Levels of these compounds are significantly increased in the temporal and parietal cortices obtained from patients with Alzheimer's disease compared with control subjects (11Reich E.E. Markesbery W.R. Roberts II, L.J. Swift L.L. Morrow J.D. Montine T.J. Am. J. Pathol. 2001; 158: 293-297Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar). DHA is concentrated in neurons, and we have thus suggested that NP formation may be a useful marker of selective neuronal oxidative injury. It is well known that eicosanoids containing E-type and D-type prostane rings are unstable and readily dehydrate in aqueous solutions to cyclopentenone-containing compounds (12Fitzpatrick F.A. Wynalda M.A. J. Biol. Chem. 1983; 258: 11713-11718Abstract Full Text PDF PubMed Google Scholar). For example PGE2and PGD2 can dehydrate to PGA2 and PGJ2, respectively. PGJ2 can undergo further metabolism to Δ12-PGJ2 and 15-deoxy-Δ12,14-PGJ2. Cyclopentenone PGs have attracted considerable interest because of their ability to modulate cell proliferation and differentiation. For example, various compounds of the A- and J-series have been shown to inhibit proliferation with a G1 cell cycle arrest and to induce differentiation (13Narumiya S. Ohno K. Fujiwara M. Fukushima M. J. Pharmacol. Exp. Ther. 1986; 239: 506-511PubMed Google Scholar, 14Cuzzocrea S. Wayman N.S. Mazzon E. Dugo L., Di Paola R. Serraino I. Britti D. Chatterjee P.K. Caputi A.P. Thiemermann C. Mol. Pharmacol. 2002; 61: 997-1007Crossref PubMed Scopus (113) Google Scholar, 15Fukushima M. Prostaglandins Leukot. Essent. Fatty Acids. 1992; 47: 1-12Abstract Full Text PDF PubMed Scopus (187) Google Scholar). The concentrations of compounds required for this activity are frequently in the micromolar range or above. On the other hand, at concentrations in the nanomolar range, A- and J-series PGs induce proliferation (16Chinery R. Coffey R.J. Graves-Deal R. Kirkland S.C. Sanchez S.C. Zackert W.E. Oates J.A. Morrow J.D. Cancer Res. 1999; 59: 2739-2746PubMed Google Scholar, 17Shahabi N.A. Chengini N. Wittliff J.L. Exp. Cell Biol. 1987; 55: 18-27PubMed Google Scholar, 18Clay C.E. Namen A.M. Atsumi G. Trimboli A.J. Fonteh A.N. High K.P. Chilton F.H. J. Invest. Med. 2001; 49: 413-420Crossref PubMed Scopus (63) Google Scholar). One limitation of the studies reported to date with cyclopentenone PGs is that there is marked paucity of evidence that they are formed in vivo (19Hirata Y. Hayashi H. Ito S. Kikawa Y. Ishibashi M. Sudo H. Miyazaki M. Fukushima S. Narumiya S. Hayaishi O. J. Biol. Chem. 1988; 263: 16619-16625Abstract Full Text PDF PubMed Google Scholar, 20Shibata T. Kondo M. Osawa T. Shibata N. Kobayashi M. Uchida K. J. Biol. Chem. 2002; 277: 10459-10466Abstract Full Text Full Text PDF PubMed Scopus (356) Google Scholar). Previously, we reported that analogous to the formation of A-ring and J-ring PGs from the dehydration of cyclooxygenase-generated PGE2 and PGD2, A2/J2-IsoPs are generated in vitroand in vivo from IsoPs containing E-type and D-type prostane rings (E2/D2-IsoPs) (21Chen Y. Morrow J.D. Roberts L.J. J. Biol. Chem. 1999; 274: 10863-10868Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar). The biological relevance of this observation relates to the fact that A2/J2-IsoPs contain α,β-unsaturated carbonyl moieties and are thus highly reactive and readily adduct, via Michael addition, GSH and protein thiols. Indeed, we have shown that in the presence of GSH and cellular GSH transferase, the A-ring IsoP 15-A2t-IsoP (8-iso-PGA2) rapidly conjugates GSH (21Chen Y. Morrow J.D. Roberts L.J. J. Biol. Chem. 1999; 274: 10863-10868Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar). The adduction of relevant reducing substances and proteins by endogenously generated IsoPs may be responsible for some of the adverse effects of oxidant stress in vivo. Analogous to the formation of A2/J2-IsoPs from the dehydration of E2/D2-IsoPs, it would be postulated that E4/D4-NPs can undergo dehydration resulting in the generation of cyclopentenone NPs, termed A4/J4-NPs. The mechanism of formation of A4/J4-NPs from DHA is shown in Fig.1, A–C. Initially, five DHA radicals are generated, and following the addition of molecular oxygen, eight peroxyl radicals result. Subsequently, the peroxyl radicals undergo endocyclization followed by addition of molecular oxygen to form eight bicyclic endoperoxide intermediate regioisomers (not shown). These regioisomers then undergo rearrangement to generate eight E4-NP and eight D4-NP regioisomers. Each regioisomer is theoretically composed of eight racemic diastereoisomers for a total of 256 E-ring and D-ring compounds. These molecules subsequently undergo dehydration to A-ring and J-ring compounds. Loss of one chiral center reduces the total number of potential A4/J4-NPs to 128. The Eicosanoid Nomenclature Committee has established and approved a nomenclature system for the IsoPs, in which the different regioisomer classes are designated by the carbon number of the side chain where the hydroxyl is located, with the carbonyl carbon designated as C1 (22Taber D.F. Morrow J.D. Roberts II, L.J. Prostaglandins. 1997; 53: 63-67Crossref PubMed Scopus (169) Google Scholar). By applying this system to NPs, A-ring and J-ring regioisomers can be designated as the 4-series A4/J4 NPs, the 7-series A4/J4-NPs, 11-series, etc. This is comparable with the series obtained with D4/E4 NPs (3Reich E.E. Zackert W.E. Brame C.J. Chen Y. Roberts II, L.J. Hachey D.L. Montine T.J. Morrow J.D. Biochemistry. 2000; 39: 2376-2383Crossref PubMed Scopus (68) Google Scholar). Here we present evidence that A4/J4-NPs are, in fact, formed in significant amounts in vitro and in vivo from the free radical catalyzed peroxidation of DHA. Docosahexaenoic acid, dimethylformamide, and undecane were purchased from Aldrich. Pentafluorobenzyl (PFB) bromide, methoxyamine HCl, and diisopropylethylamine were from Sigma. 2,2′-Azobis-(2-amidinopropane) hydrochloride (AAPH) was from Eastman Kodak Co. [2H3]Methoxyamine HCl was from Cambridge Isotope Laboratories, Inc. (Andover, MA).N,O-Bis(trimethylsilyl)trifluoroacetamide (BSTFA) was from Supelco (Bellefonte, PA). [2H9]N,O-Bis(trimethylsilyl)acetamide was from CDN Isotopes (Pointe-Claire, Quebec, Canada). C-18 and Silica Sep-Pak cartridges were from Waters Associates. 60ALK6D TLC plates were from Whatman. [2H4]PGA2 and [2H4]PGE2 were from Cayman Biochemicals (Ann Arbor, MI). B & J Inert SPE System Columns (Glass Sep-Paks) were from Burdick and Jackson (Muskegon, MI). DHA was oxidized in vitro using an iron/ADP/ascorbate mixture, as described previously (23Longmire A.J. Swift L.L. Roberts II, L.J. Awad J.A. Burke R.F. Morrow J.D. Biochem. Pharmacol. 1994; 47: 1173-1177Crossref PubMed Scopus (121) Google Scholar). Samples obtained from oxidation of DHA or from other sources were extracted in ethyl acetate and subsequently converted to O-methyloxime derivatives by incubation with methoxyamine HCl in pyridine for 45 min at 55 °C. A4/J4 NPs were then extracted using C-18 Sep-Pak cartridges as described (3Reich E.E. Zackert W.E. Brame C.J. Chen Y. Roberts II, L.J. Hachey D.L. Montine T.J. Morrow J.D. Biochemistry. 2000; 39: 2376-2383Crossref PubMed Scopus (68) Google Scholar, 21Chen Y. Morrow J.D. Roberts L.J. J. Biol. Chem. 1999; 274: 10863-10868Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar). The eluted samples were converted to O-methyloxime PFB ester derivatives, purified by TLC using 60ALK6D silica gel plates employing a solvent system of hexane/acetone (70:30, v/v) as utilized previously for A2/J2-IsoPs (21Chen Y. Morrow J.D. Roberts L.J. J. Biol. Chem. 1999; 274: 10863-10868Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar). Compounds migrating in the region from 2 cm below to 1.5 cm above the methyloxime PFB ester of PGA2 (R f = 0.27–0.54) were scraped, extracted with ethyl acetate, converted to trimethylsilyl (TMS) ether derivatives, and quantified by stable isotope dilution techniques employing gas chromatography (GC)/negative ion chemical ionization (NICI)/mass spectrometry (MS). The standard used for quantification of A4/J4-NPs was [2H4]PGA2. The major ions generated in the NICI mass spectra of the PFB ester,O-methyloxime, TMS ether derivatives of A4/J4-NPs and the [2H4]PGA2 are the carboxylate anions at m/z 458 and m/z438, respectively. Quantification of A4/J4-NPs was performed based on integration of peak areas. GC/NICI/MS was carried out using either a Hewlett-Packard 5890 GC/MS (Palo Alto, CA) or a Thermo-Finnigan Voyager GC/MS (San Jose, CA). Treatment of PGA2 with BSTFA and piperidine has been shown to convert it to a piperidyl-enol-TMS ether derivative, which is specific for A-ring prostanoids (21Chen Y. Morrow J.D. Roberts L.J. J. Biol. Chem. 1999; 274: 10863-10868Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar). Thus, we analyzed for the formation of this derivative of A4/J4-NPs. PFB esters of putative cyclopentenone-NPs were treated with a 1:1 mixture of BSTFA/piperidine for 1 h at 60 °C and analyzed by GC/NICI/MS monitoring the carboxylate anions at m/z 586 for the NPs andm/z 566 for [2H4]PGA2. In some experiments, compounds were exposed to catalytic hydrogenation after conversion to piperidyl-enol-TMS ether derivatives as described (24Morrow J.D. Harris T.M. Roberts L.J. Anal. Biochem. 1990; 184: 1-10Crossref PubMed Scopus (410) Google Scholar). Compounds generated by in vitrooxidation of DHA were purified by normal phase HPLC using a 25 cm × 4.6 mm Econosil SI column with 5-μm particles employing an isocratic solvent system of hexane/isopropyl alcohol/acetic acid (97:3:0.1, v/v/v) at a flow rate of 1 ml/min. A4/J4-NPs eluted in retention volumes 13–37 ml. Fractions were analyzed by GC/MS for A4/J4-NPs. Selected fractions that contained substantial amounts of A4/J4-NPs were acidified with 1 n HCl, extracted with 2 volumes of ethyl acetate, dried, and redissolved in a small volume of ethanol. Fractions were then analyzed by LC/ESI/MS/MS in the negative ion mode using a 5 cm × 2.1 mm Zorbax C-18 column (Agilent Technologies, Palo Alto, CA). The solvent system was a gradient consisting of 5 mmammonium acetate/acetonitrile/acetic acid (90:10:0.1, v/v/v) to 5 mm ammonium acetate/acetonitrile/acetic acid (10:90:0.1, v/v/v) over the course of 10 min at a flow rate of 200 μl/min. The auxiliary gas pressure was 10 liters/min, and the sheath gas pressure was 60 lb/in2. The voltage on the capillary was 20.0 V; the capillary temperature was 200 °C, and the tube lens voltage was 75.0 V. Parent ions were scanned from m/z 300 to 400. Collision-induced dissociation (CID) of molecular ions of putative A4/J4-NPs in these fractions was performed from 20 to 30 eV scanning daughter ions from m/z 50 to 400. The spectrum shown was obtained at 25 eV. The CID gas was argon with a pressure set at 2.5 millitorr. Spectra were displayed by averaging scans across chromatographic peaks. LC/MS was carried out using a Finnigan TSQ 7000 instrument (San Jose, CA). Brain tissue was obtained from male Sprague-Dawley rats and homogenized, and synaptosomes were isolated by Ficoll gradient centrifugation (25Blakely R.D. Ory-Lavollee L. Thopmson R.C. Coyle J.T. J. Neurochem. 1986; 47: 1013-1019Crossref PubMed Scopus (37) Google Scholar). Lipid peroxidation was initiated by the addition of AAPH (final concentration of 5 mm). Incubations were carried out at 37 °C for 2 h. The reactions were terminated by placing the samples at −80 °C, and lipids were extracted by the method of Folch (see Ref. 3Reich E.E. Zackert W.E. Brame C.J. Chen Y. Roberts II, L.J. Hachey D.L. Montine T.J. Morrow J.D. Biochemistry. 2000; 39: 2376-2383Crossref PubMed Scopus (68) Google Scholar). Esterified A4/J4-NPs in phospholipids were hydrolyzed using chemical saponification with potassium hydroxide (26Morrow J.D. Roberts II, L.J. Daniel V.C. Awad J.A. Mirochnitchenko O. Swift L.L. Burke R.F. Arch. Biochem. Biophys. 1998; 353: 160-171Crossref PubMed Scopus (74) Google Scholar), following treatment with methoxyamine HCl in chloroform/methanol 2:1 for 1 h at room temperature. Purification was performed as for free A4/J4-NPs, and levels of A4/J4-NPs were then quantified by GC/MS. Data are expressed relative to protein concentration as determined by the Pierce BCA assay (3Reich E.E. Zackert W.E. Brame C.J. Chen Y. Roberts II, L.J. Hachey D.L. Montine T.J. Morrow J.D. Biochemistry. 2000; 39: 2376-2383Crossref PubMed Scopus (68) Google Scholar). Tissue samples were obtained from adult male Sprague-Dawley rats and postmortem human parietal lobe tissue (informed consent obtained). Samples were homogenized in chloroform/methanol 2:1 containing BHT (0.005%) and triphenylphosphine (10 mg/ml) to prevent autoxidation. 2 ml of 0.9% NaCl were added to the samples; they were then shaken and centrifuged, and the aqueous layer was discarded. The samples were dried under nitrogen and resuspended in 1 ml of chloroform, and phospholipids containing A4/J4-NPs were separated from other lipids as follows. A glass silica Sep-Pak (500 mg) was prewashed with 5 ml of hexane followed by 5 ml of chloroform. Each sample was applied to the Sep-Pak and initially extracted with 12 ml of hexane/MTBE (200:3, v/v). Unoxidized cholesterol esters and triglycerides elute in this fraction. Subsequently, the column was extracted with 12 ml of methanol/MTBE (5:95, v/v) to remove oxidized non-polar lipids and cholesterol. A final extraction using 15 ml of MTBE/methanol/ammonium acetate (0.0 1m) (5:8:2 v/v/v) elutes oxidized and unoxidized phospholipids. In preliminary studies, >98% of A4/J4-NPs eluted in the phospholipid fraction. Further purification of A4/J4-NPs was identical to that employed for the rat synaptosomes as noted above. A4/J4-NPs (∼100 ng) obtained from the in vitro oxidation of DHA were purified by Sep-Pak extraction and TLC and incubated in 0.1 m KPO4buffer (pH 6.5) in the presence of a 10-fold excess (∼1 μg) of GSH and 1 mg of bovine liver GSH transferase (Sigma) containing a mixture of GSH transferases at 37 °C for 2 h (21Chen Y. Morrow J.D. Roberts L.J. J. Biol. Chem. 1999; 274: 10863-10868Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar). The incubation mixture was then acidified to pH 3 and extracted with 2 volumes of MeCl2. Unconjugated A4/J4-NPs were measured in the organic fraction by GC/MS, and conjugated NPs represented the difference between the amount of A4/J4-NPs added to the incubationversus that amount present in the organic fraction. Subsequently, A4/J4-NP-GSH adducts from a separate incubation were definitively identified by LC/MS. Adducts were purified by extraction using a C18 Sep-Pak cartridge preconditioned with acetonitrile and 0.1 m ammonium acetate (pH 3.4) (21Chen Y. Morrow J.D. Roberts L.J. J. Biol. Chem. 1999; 274: 10863-10868Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar) and eluted with 10 ml of 95% ethanol and analyzed by LC/MS. LC was carried out using a MAGIC 2002 LC system (Michrom BioResources, Auburn, CA) operating in the isocratic mode with the mobile phase of H2O/acetonitrile/acetic acid (77:22.9:0.1, v/v/v), and compounds were separated on an Eclipse XDB-C18 column (2.1 × 50 mm, 5-μm particle size; Agilent, Palo Alto, CA) at a flow rate of 75 μl/min. Following on-line chromatography, samples were characterized employing a Finnigan TSQ-7000 (San Jose, CA) triple quadripole mass spectrometer operating in the positive ion mode. An electrospray source was fitted with a 100 μm internal diameter deactivated fused silica capillary column and used nitrogen for both sheath and auxiliary gas, operating at 60 pounds/square inch and 10 liters/min, respectively. The ESI potential was maintained at 3.5 kV, the heated capillary at 20 V and 200 °C, and the tube lens 70 V. For tandem MS, parent compounds were collisionally activated at an energy of −15 eV and under 2.5 millitorrs of argon. Data acquisition and analysis were performed using an Alpha work station (Digital Equipment Corp., Maynard, MA) running Finnigan ICIS software, version 8.3.2. A representative selected ion current chromatogram obtained from the oxidation of DHA in vitro with iron/ADP/ascorbate for 6 h is shown in Fig. 2. The two large chromatographic peaks shown in the m/z 438 channel represent the syn- and anti-O-methyloxime isomers of the internal standard [2H4]PGA2. In the upperm/z 458 ion current chromatograms are a series of chromatographic peaks eluting over approximately a 2-min interval. These compounds possessed a molecular mass predicted for A4/J4-NPs. In addition, it would be predicted that the retention time of A4/J4-NPs on GC should be longer than that of the deuterated PGA2 internal standard because the former compounds contain two additional carbon atoms (3Reich E.E. Zackert W.E. Brame C.J. Chen Y. Roberts II, L.J. Hachey D.L. Montine T.J. Morrow J.D. Biochemistry. 2000; 39: 2376-2383Crossref PubMed Scopus (68) Google Scholar). As for PGA2 and PGJ2, as well as for the A2/J2-IsoPs, that have similar chromatographic properties on TLC and GC and identical molecular masses, it is not possible to differentiate between A-type and J-type prostane rings in the putative NP compounds detected in them/z 458 ion current chromatogram shown in Fig. 2. However, because both PGE2 and PGD2 readily dehydrate in aqueous solutions to form PGA2 and PGJ2, respectively, it is expected that the D4-NPs and E4-NPs would dehydrate to form A4-NPs and J4-NPs. Additional experimental approaches were undertaken to provide further evidence that the compounds represented by the chromatographic peaks in the m/z 458 ion current chromatogram were A4/J4-NPs. First, the m/z457 ion current chromatogram contained no chromatographic peaks, indicating that the peaks in the m/z 458 chromatogram are not natural isotope peaks of compounds generating an ion of less than m/z 458. Analysis of putative A4/J4-NPs as [2H9]TMS ether derivatives resulted in a shift of the m/z458 chromatographic peaks up 9 Da to m/z 467, indicating the presence of 1 hydroxyl group (data not shown). When these compounds were analyzed asO-[2H3]methyloxime derivatives, the m/z 458 chromatographic peaks all shifted up to m/z 461, indicating the presence of 1 carbonyl group (not shown). Subsequently, analysis following treatment with BSTFA/piperidine resulted in the formation of piperidyl-enol-TMS ether derivatives (Fig. 3 A). The amount of A4/J4-NPs analyzed as this derivative was calculated to be 547 ± 232 ng/mg DHA (mean ± S.E.,n = 3), which is less than the amount formed when compounds are analyzed as O-methyloxime, TMS ether derivatives. This discrepancy can be explained by the fact that we have found that, although treatment of PGA2 with BSTFA/piperidine efficiently converts it to a piperidyl-enol-TMS ether derivative, only small amounts of this derivative are formed with PGJ2. By analogy, therefore, only a portion of the mixture of A4/J4-NPs would be expected to form a piperidyl-enol-TMS ether derivative. Finally, putative A4/J4-NPs were analyzed following catalytic hydrogenation as piperidyl-enol-TMS ether derivatives. Prior to hydrogenation, there were no chromatographic peaks present 10 Da above the m/z 586 in the m/z 596 chromatogram. However, following hydrogenation, intense chromatographic peaks appeared at m/z 596 (Fig. 3 B) with the loss of chromatographic peaks at m/z586, indicating the presence of 5 double bonds. It should be noted that it was not possible to analyze putative A4/J4-NPs following hydrogenation asO-methyloxime TMS ether derivatives because of the presence of interfering chromatographic peaks 10 Da abovem/z 458. Collectively, these data indicated that the compounds represented by the chromatographic peaks in them/z 458 ion current chromatogram shown in Fig. 2have the functional groups and the number of double bonds predicted for A4/J4-NPs. To provide direct evidence that the compounds analyzed by selected ion monitoring MS were A4/J4-NPs, LC/ESI/MS/MS in the negative ion mode was employed. The material was purified before LC/MS analysis by HPLC, and eluted fractions containing significant amounts of putative A4/J4-NPs as determined by GC/MS were then analyzed by LC/MS. Notably, A4/J4-NPs eluted over a very broad volume from 13 to 37 ml using this HPLC solvent system (Fig.4 A). Subsequently, 1-ml fractions were analyzed by LC/MS. The predicted [parent molecule − H]− ion, hereafter referred to as [M − H]−, for A4/J4-NPs ism/z 357. Fig. 4 B shows the selected ion monitoring chromatogram of the ion at m/z 357 obtained from one of the analyses of putative cyclopentenone NPs eluting from the HPLC at 18 ml. In this fraction, A4/J4-NPs elute from the LC column as a relatively broad set of peaks over about 1 min. A composite collision-induced dissociation (CID) spectrum obtained by summing scans over the broad chromatographic peak in Fig. 4 B is shown in Fig. 4 C. CID of the ion at m/z 357 resulted in the formation of a number of relevant daughter ions that would be predicted to be common to all of the A4/J4-NPs, including m/z339 ([M − H] − H2O)−,m/z 313 ([M − H] − CO2)−, and m/z 295 ([M − H] − H2O − CO2)−. Other prominent daughter ions are present that may result from fragmentation of specific A4/J4-NP regioisomers. Because of the limited amount of material and the lack of chemically synthesized A- and J-ring NPs, it is impossible to know with certainty the chemical structures of these smaller fragments. Nonetheless, on the basis of our previous work (3Reich E.E. Zackert W.E. Brame C.J. Chen Y. Roberts II, L.J. Hachey D.L. Montine T.J. Morrow J.D. Biochemistry. 2000; 39: 2376-2383Crossref PubMed Scopus (68) Google Scholar) and studies by Kerwin et al. (27Kerwin J.L. Torvik J.J. Anal. Biochem. 1996; 237: 56-64Crossref PubMed Scopus (60) Google Scholar, 28Kerwin J.L. Wiens A.M. Ericssom L.H. J. Mass Spectrom. 1996; 31: 184-192Crossref PubMed Scopus (123) Google Scholar), Oliwet al. (29Oliw E.H., Su, C. Skogstrom T. Benthin G. Lipids. 1998; 33: 843-852Crossref PubMed Scopus (67) Google Scholar), and Murphy and co-workers (30Waugh R.J. Morrow J.D. Roberts II, L.J. Murphy R.C. Free Radic. Biol. Med. 1997; 23: 943-954Crossref PubMed Scopus (113) Google Scholar, 31Zirolli J.A. Davoli E. Bettazzoli L. Gross M. Murphy R.C. J. Am. Soc. Mass Spectrom. 1990; 1: 325-335Crossref PubMed Scopus (53) Google Scholar), chara" @default.
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• W2114305841 title "Formation of Highly Reactive A-ring and J-ring Isoprostane-like Compounds (A4/J4-neuroprostanes) in Vivo from Docosahexaenoic Acid" @default.
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