Intramolecular rearrangement of linolenate peroxyl radicals in lipoxygenase reactions at lower oxygen content

Variation of tissue oxygen content is thought to be a possible factor in determining the structural diversity of hydroperoxy fatty acids. In the present study, we evaluated the structural diversity of intermediate carbon-centered radicals at lower oxygen content. When the buffered solution (pH 7.4) containing 1.0 mM α-linolenic acid, 1.0 μM soybean 15-lipoxygenase, and 1.0 mM nitroxyl radical [3-carbamoyl-2,2,5,5-tetramethyl-3-pyrroline-N-oxyl (CmΔP)], which selectively traps carbon-centered radicals, was incubated in a sealed vial, the generation of linolenate hydroperoxide was completed within 1 min. In the subsequent reaction at lower oxygen content, the production of the [LnA−H+O2]·-CmΔP adduct was ascertained by liquid chromatography tandem mass spectrometry with precursor ion scanning. Furthermore, HPLC analysis with photodiode array detection showed that the adduct exhibits an absorption maximum at 278 nm, indicating a conjugated triene moiety. On the basis of these facts, the structure of the adduct was speculated to be C2H5−CH(CmΔP)−CH = CH−CH = CH−CH = CH−CH(OOH) −C7H14−COOH. We proposed a possible reaction pathway as follows: a linolenate 9-peroxyl radical generated in the lipoxygenase reaction might be converted into C2H5−·CH−CH = CH−CH = CH−CH = CH−CH(OOH) −C7H14−COOH through an intramolecular rearrangement. This intermediate radical may give rise to hydroperoxy fatty acids with structural diversity. Variation of tissue oxygen content is thought to be a possible factor in determining the structural diversity of hydroperoxy fatty acids. In the present study, we evaluated the structural diversity of intermediate carbon-centered radicals at lower oxygen content. When the buffered solution (pH 7.4) containing 1.0 mM α-linolenic acid, 1.0 μM soybean 15-lipoxygenase, and 1.0 mM nitroxyl radical [3-carbamoyl-2,2,5,5-tetramethyl-3-pyrroline-N-oxyl (CmΔP)], which selectively traps carbon-centered radicals, was incubated in a sealed vial, the generation of linolenate hydroperoxide was completed within 1 min. In the subsequent reaction at lower oxygen content, the production of the [LnA−H+O2]·-CmΔP adduct was ascertained by liquid chromatography tandem mass spectrometry with precursor ion scanning. Furthermore, HPLC analysis with photodiode array detection showed that the adduct exhibits an absorption maximum at 278 nm, indicating a conjugated triene moiety. On the basis of these facts, the structure of the adduct was speculated to be C2H5−CH(CmΔP)−CH = CH−CH = CH−CH = CH−CH(OOH) −C7H14−COOH. We proposed a possible reaction pathway as follows: a linolenate 9-peroxyl radical generated in the lipoxygenase reaction might be converted into C2H5−·CH−CH = CH−CH = CH−CH = CH−CH(OOH) −C7H14−COOH through an intramolecular rearrangement. This intermediate radical may give rise to hydroperoxy fatty acids with structural diversity. It is generally accepted that lipoxygenase, in combination with phospholipase A2, is involved in ischemia-reperfusion injury (1Arai K. Nishiyama N. Matsuki N. Ikegaya Y. Neuroprotective effects of lipoxygenase inhibitors against ischemic injury in rat hippocampal slice cultures. Brain Res. 2001; 904: 167-172Google Scholar, 2Phillis J.W. O'Regan M.H. The role of phospholipases, cyclooxygenases, and lipoxygenases in cerebral ischemic/traumatic injuries. Crit. Rev. Neurobiol. 2003; 15: 61-90Crossref PubMed Scopus (85) Google Scholar, 3Patel N.S. Cuzzocrea S. Chatterjee P.K. Di Paola R. Sautebin L. Britti D. Thiemermann C. Reduction of renal ischemia-reperfusion injury in 5-lipoxygenase knockout mice and by the 5-lipoxygenase inhibitor zileuton. Mol. Pharmacol. 2004; 66: 220-227Google Scholar, 4Cummings B.S. Mchowat J. Schnellmann R.G. Phospholipase A2s in cell injury and death. J. Pharmacol. Exp. Ther. 2000; 294: 793-799PubMed Google Scholar, 5Sapirstein A. Bonventre J.V. Phospholipase A2 in ischemic and toxic brain injury. Neurochem. Res. 2000; 25: 745-753Crossref PubMed Scopus (66) Google Scholar, 6Sun G.Y. Xu J. Jensen M.D. Simonyi A. Phospholipase A2 in the central nervous system: implications for neurodegenerative diseases. J. Lipid Res. 2004; 45: 205-213Google Scholar). However, the real mechanism of how the tissue is injured via lipoxygenase and polyunsaturated fatty acids (PUFAs) remains obscure. Furthermore, there are few examples of structural diversity in the resulting hydroperoxy fatty acids and the reaction pathway under ischemic conditions. Lipoxygenases insert an oxygen molecule into an individual position in PUFAs including arachidonic acid, eicosapentaenoic acid, linoleic acid, and α-linolenic acid. 15-Lipoxygenase and 5-lipoxygenase are the principal isozymes involved in inflammatory responses. At the initial stage of lipid peroxidation by lipoxygenase, a doubly allylic hydrogen should be abstracted from PUFA, which is accompanied by reduction of ferric lipoxygenase to the ferrous form. It seems that an oxygen molecule can reach the carbon-centered radicals through a channel in the lipoxygenase, resulting in the highly controlled stereo- and regiospecificities of the resultant hydroperoxy fatty acid (7Knapp M.J. Klinman J.P. Kinetic studies of oxygen reactivity in soybean lipoxygenase-1. Biochemistry. 2003; 42: 11466-11475Google Scholar). In general, decreased oxygen content results in a lack of regiospecificity (8Berry H. Débat H. Garde V.L. Oxygen concentration determines regiospecificity in soybean lipoxygenase-1 reaction via a branched kinetic scheme. J. Biol. Chem. 1998; 273: 2769-2776Google Scholar). In fact, it was demonstrated that 15-lipoxygenase inserts an oxygen molecule into linoleic acid and α-linolenic acid at the C-9 position as well as the C-13 position at lower oxygen content (9Ivanov I. Rathmann J. Myagkova G. Kuhn H. Soybean lipoxygenase-1 oxygenates synthetic polyenoic fatty acids with an altered positional specificity. Evidence for inverse substrate alignment. Biochemistry. 2001; 40: 10223-10229Google Scholar, 10Gardner H.W. Soybean lipoxygenase-1 enzymatically forms both (9S)- and (13S)-hydroperoxides from linoleic acid by a pH-dependent mechanism. Biochim. Biophys. Acta. 1989; 1001: 274-281Google Scholar, 11Nuñez A. Foglia T.A. Piazza G.J. Characterization of lipoxygenase oxidation products by high-performance liquid chromatography with electron impact-mass spectrometric detection. Lipids. 2001; 36: 851-856Google Scholar). This fact indicates that the binding affinity of lipid allyl radicals to the lipoxygenase should be loose at lower oxygen content as compared with that in normoxia. In order to evaluate the structural diversity of hydroperoxy fatty acids produced in the PUFA/lipoxygenase system at lower oxygen content, it is effective to evaluate the generation of the fatty acid-derived carbon-centered radicals with which the oxygen molecule can react. Furthermore, the evaluation of the intermediate carbon-centered radical generation could lead to the elucidation of the reaction pathway. For this purpose, we established a novel method for the detection of fatty acid-derived carbon-centered radicals via the combination of the nitroxyl radical spin-trapping technique with the precursor ion-scanning (PIS) technique in liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis. The nitroxyl radical, 3-carbamoyl-2,2,5,5-tetramethyl-3-pyrroline-N-oxyl (CmΔP), was employed to selectively trap carbon-centered radicals, although the resulting adducts are comparatively thermolabile (12Koshiishi I. Tsuchida K. Takajo T. Komatsu M. Quantification of lipid alkyl radicals trapped with nitroxyl radical via HPLC with postcolumn thermal decomposition. J. Lipid Res. 2005; 46: 2506-2513Google Scholar). In the present study, we set up an in vitro hypoxia condition with soybean 15-lipoxygenase and α-linolenic acid: Oxygen was consumed by the dioxygenation of PUFA in the lipoxygenase reaction. The fatty acid-derived carbon-centered radical-CmΔP adducts produced in the linolenate/lipoxygenase system in the presence of CmΔP were identified by LC-MS/MS with PIS and HPLC with photodiode array detection. And then, on the basis of the adduct structure, we speculated a possible pathway for fatty acid-derived carbon-centered radical generation at lower oxygen content. Soybean lipoxygenase-1 (type I-b; activity, 70,800 U/mg; molecular mass, 108 kDa), linoleic acid, and α-linolenic acid were purchased from Sigma. 13-Hydroperoxy-(9Z,11E)-octadecadienoic acid was purchased from Wako Pure Chemical Industries Ltd. (Osaka, Japan). CmΔP was purchased from Aldrich Chemical Co., Inc. and was recrystallized in ethanol before use. TSKgel ODS-80Ts QA and TSKguardgel ODS-80Ts were purchased from Tosoh Co. (Tokyo, Japan). Chelex® 100 Resin (100–200 mesh) was purchased from Bio-Rad. All other chemicals used were reagent grade. If necessary, α-linolenic acid was chromatographically purified as follows: 2 ml of 50 mM α-linolenic acid in 50% acetonitrile was passed through two Sep-Pak Plus C18 columns (Waters Co.). α-Linolenic acid on the columns was eluted by water-acetonitrile gradient elution. These operations were performed in a nitrogen atmosphere. Contaminant linolenate hydroperoxide was eluted in front of α-linolenic acid. The α-linolenic acid fraction was evaporated, and the residue was dissolved in ethanol. The concentration was adjusted to 100 mM, and the solution was stored at −80°C. Lipid-derived radicals generated in the PUFA/lipoxygenase system were trapped with the nitroxyl radical CmΔP as follows: 20 μl of 2 mM PUFA emulsion in 0.1 M phosphate buffer (pH 7.4, treated with Chelex 100) containing 2% ethanol was mixed with 10 μl of 4 mM nitroxyl radical in 0.1 M phosphate buffer (pH 7.4, treated with Chelex 100) and 10 μl of 4 μM soybean lipoxygenase-1 in 0.1 M phosphate buffer (pH 7.4, treated with Chelex 100) in a glass vial tube with screw cap (inner volume, 0.5 ml), which was incubated at 25°C for 5 min. The reaction solution was mixed with 160 μl of cold acetonitrile and then centrifuged at 10,000 g for 1 min at 0°C. The supernatant was subjected to HPLC. The chromatographic conditions for the quantification of the lipid-derived radical-nitroxyl radical adducts are as follows: column, TSKgel ODS-80Ts QA (4.6 mm i.d. × 150 mm) with guard column, TSKguardgel ODS-80Ts (3.2 mm i.d. × 15 mm); eluent, 75% acetonitrile containing 0.05% formic acid; flow rate, 1.0 ml/min; column temperature, 25–28°C. The on-line LC-MS/MS system consisted of the Agilent1100 HPLC system and API 4000® Triple Quadrupole LC-MS/MS system (Applied Biosystems/MDS Sciex; Concord, ON, Canada) equipped with an electrospray ion source. Tandem mass spectrometry (MS/MS) conditions for API-4000 are as follows: polarity, positive; curtain gas, 50 psi; ion source gas 1, 30 psi; ion source gas 2, 70 psi; ion spray voltage, 5,500 V; temperature, 600°C; collision gas, 1.00; declustering potential, 81 V; entrance potential, 10 V; collision cell exit potential, 15 V; collision energy, 30 V; channel electron multiplier, 2,000 V; deflector, −100 V. The HPLC with photodiode array detection system consisted of a pump (LC-20AD; Shimadzu, Japan), a degasser (DGU-20A5; Shimadzu), a photodiode array detector (GL-7452; GL Science, Japan), a column oven (CTO-20A; Shimadzu), and an auto-sampler (SIL-20A, Shimadzu). The chromatographic conditions for the quantification of the PUFAs and hydroperoxides were the same as those for the quantification of the lipid-derived radical-nitroxyl radical adducts. PUFAs and hydroperoxides were detected at 210 nm and 234 nm, respectively. Lipid-derived radicals generated in the linolenate/lipoxygenase system in the presence of 13-hydroperoxy-octadecadienoic acid were trapped with the nitroxyl radical CmΔP as follows: 10 μl of 4 mM α-linolenic acid emulsion in 0.1 M phosphate buffer (pH 7.4, treated with Chelex 100) containing 4% ethanol was mixed with 10 μl of 13-hydroperoxy-octadecadienoic acid in 0.1 M phosphate buffer (pH 7.4, treated with Chelex 100), 10 μl of 4 mM nitroxyl radical in 0.1 M phosphate buffer (pH 7.4, treated with Chelex 100), and 10 μl of 4 μM soybean lipoxygenase-1 in 0.1 M phosphate buffer (pH 7.4, treated with Chelex 100) in a glass vial tube with screw cap (inner volume, 0.5 ml), which was incubated at 25°C for 5 min. The reaction solution was mixed with 160 μl of cold acetonitrile and then centrifuged at 10,000 g for 1 min at 0°C. The supernatant was subjected to HPLC with ultraviolet (UV) detection. In the present study, soybean lipoxygenase-1 (type I-b) and α-linolenic acid were employed as 15-lipoxygenase and PUFA, respectively, to evaluate the generation of fatty acid-derived carbon-centered radicals in a lipoxygenase reaction at lower oxygen content. To establish the lower oxygen condition, we employed a linolenate/lipoxygenase system, in which dissolved oxygen (approximately 230 μM) is consumed by the dioxygenation of α-linolenic acid. When the phosphate buffer solution (0.1 M, pH 7.4) containing 1.0 mM α-linolenic acid and 1.0 μM lipoxygenase was incubated in a sealed vial at 25°C, the linolenate hydroperoxide content reached a plateau within 1 min (data not shown). To detect lipid-derived carbon-centered radicals, it is essential for the spin-trapping agent to trap every type of carbon-centered radical. In our recent study (12Koshiishi I. Tsuchida K. Takajo T. Komatsu M. Quantification of lipid alkyl radicals trapped with nitroxyl radical via HPLC with postcolumn thermal decomposition. J. Lipid Res. 2005; 46: 2506-2513Google Scholar), we demonstrated that the nitroxyl radical, CmΔP: M.W., 183, stoichiometrically trapped lipid-derived carbon-centered radicals, including lipid allyl radicals and lipid epoxyallyl radicals, in the linoleate/lipoxygenase system through radical-radical conjunction. We confirmed that the presence of the nitroxyl radical did not prevent the lipoxygenase reaction as follows: when 1.0 mM linoleate was incubated with 0.1 μM lipoxygenase in the presence or absence of CmΔP (1.0 mM), there is no significant difference in the initial rate of hydroperoxide generation between the two systems (approximately 2.4 μM/sec). Remarkably, the nitroxyl radical scavenges the linoleate allyl radical on the ferrous lipoxygenase at lower oxygen content, resulting in the release of the inactive ferrous lipoxygenase (13Koshiishi I. Tsuchida K. Takajo T. Komatsu M. Radical scavenger can scavenge lipid allyl radicals complexed with lipoxygenase at lower oxygen content. Biochem. J. 2006; 395: 303-309Google Scholar). It is generally known that lipid hydroperoxides play a key role in the conversion of ferrous lipoxygenase into active ferric lipoxygenase, generating lipid alkoxyl radicals (14de Groot J.J.M.C. Veldink G.A. Vliegenthart J.F.G. Boldingh J. Wever R. van Gelder B.F. Demonstration by EPR spectroscopy of the functional role of iron in soybean lipoxygenase-1. Biochim. Biophys. Acta. 1975; 377: 71-79Google Scholar, 15Verhagen J. Veldink G.A. Egmond M.R. Vliegenthart J.F.G. Boldingh J. Star J.V.D. Steady-state kinetics of the anaerobic reaction of soybean lipoxygenase-1 with linoleic acid and 13-l-hydroperoxylinoleic acid. Biochim. Biophys. Acta. 1978; 529: 369-379Google Scholar, 16Schilstra M.J. Veldink G.A. Verhagen J. Vliegenthart J.F.G. Effect of lipid hydroperoxide on lipoxygenase kinetics. Biochemistry. 1992; 31: 7692-7699Google Scholar), which easily turn into epoxyallyl radicals through intramolecular rearrangement. Interestingly, MS/MS spectra of these adducts showed that carbon-centered radical-CmΔP adducts characteristically fragmented into hydroxylamine (m/z 185), which arises from the reduced form of CmΔP through heterolysis of the C-O-N bond (12Koshiishi I. Tsuchida K. Takajo T. Komatsu M. Quantification of lipid alkyl radicals trapped with nitroxyl radical via HPLC with postcolumn thermal decomposition. J. Lipid Res. 2005; 46: 2506-2513Google Scholar). This characteristic fragmentation of lipid-derived carbon-centered radical-CmΔP adducts should lead to the selective detection of adducts via MS/MS with PIS (MS/MS-PIS) for m/z 185. With this phenomenon in mind, we tried to detect nitroxyl radical spin-trapping adducts of lipid-derived carbon-centered radicals generated from linoleic acid and α-linolenic acid through the lipoxygenase reaction by using liquid chromatography mass spectrometry/mass spectrometry precursor ion scanning (LC-MS/MS-PIS). The total ion chromatograms of adducts detected by the PIS for m/z 185 are shown in Fig. 1A ,B. The cumulative mass spectra of the precursor ions that correspond to lipid-derived carbon-centered radical-CmΔP adducts eluted from 0 min to 30 min are shown in Fig. 1C, D. In both cases, lipid allyl radical-CmΔP adducts and lipid epoxyallyl radical-CmΔP adducts were detected. The scavenging of lipid allyl radicals on the ferrous lipoxygenase by the nitroxyl radical promotes the one-electron redox cycle reaction (13Koshiishi I. Tsuchida K. Takajo T. Komatsu M. Radical scavenger can scavenge lipid allyl radicals complexed with lipoxygenase at lower oxygen content. Biochem. J. 2006; 395: 303-309Google Scholar). The [LnA−H+O2]·-CmΔP adduct was detected in the linolenate/lipoxygenase/CmΔP system (Fig. 1D), whereas the [LA−H+O2]·-CmΔP adduct was not detected (Fig. 1C). It should be noted that the signal intensity does not correspond to the of adduct to the total of adducts. The ion chromatograms of carbon-centered radical-CmΔP adducts are shown in Fig. 2 Remarkably, a principal adduct to the at min was detected in the linolenate/lipoxygenase/CmΔP To the conjugated in the spectra of the [LnA−H+O2]·-CmΔP adduct (3.2 and the linolenate allyl radical-CmΔP adducts and were by photodiode array detection (Fig. both linolenate allyl radical-CmΔP adducts a at 234 nm, indicating that the linolenate allyl radical-CmΔP adducts a conjugated = CH−CH = in In the [LnA−H+O2]·-CmΔP adduct a at 278 nm, indicating that the [LnA−H+O2]·-CmΔP adduct a conjugated triene = CH−CH = CH−CH = in a carbon-centered radical to was not produced in the linoleate/lipoxygenase a carbon-centered radical to was produced in the linolenate/lipoxygenase and the production of a carbon-centered radical to was accompanied by the of a conjugated triene moiety. It should be noted we could not the adduct to structural the adduct is thermolabile that it is to (12Koshiishi I. Tsuchida K. Takajo T. Komatsu M. Quantification of lipid alkyl radicals trapped with nitroxyl radical via HPLC with postcolumn thermal decomposition. J. Lipid Res. 2005; 46: 2506-2513Google Scholar, The thermal of J. Chem. (UV) spectra of CmΔP spin-trapping adducts of carbon-centered radicals produced in the linolenate/lipoxygenase/CmΔP solution was subjected to HPLC with photodiode array detection. of CmΔP spin-trapping adducts detected at 234 nm and 278 nm, respectively. spectra of CmΔP spin-trapping adducts eluted at and a doubly allylic hydrogen from of α-linolenic acid. The intermediate carbon-centered radical into two radicals at the C-9 and C-13 In an oxygen molecule selectively the C-13 resulting in the generation of a linolenate radical. In an oxygen molecule the C-9 position as well as the C-13 position at lower oxygen content. Interestingly, CmΔP with the oxygen molecule for the linolenate allyl radical on the lipoxygenase at lower oxygen content. shown in Fig. two of adducts to the linolenate allyl adduct and the were detected by This fact indicates that linolenate 9-peroxyl radical was generated in this linolenate/lipoxygenase If α-linolenic acid is converted into a linolenate a conjugated triene two hydrogen to be abstracted from both of the doubly allylic at and in To the reaction of CmΔP with the carbon-centered radical to we speculated a mechanism as shown in Fig. 4 This reaction mechanism is on the intramolecular of the linolenate 9-peroxyl radical. In the generated principal radical is the linolenate radical, which no doubly allylic the radical on the lipoxygenase be reduced by the ferrous In the radical in the linolenate 9-peroxyl radical can a hydrogen from the doubly allylic at resulting in the of a carbon-centered radical with a conjugated triene moiety. CmΔP can with the carbon-centered radical, resulting in the of = CH−CH = CH−CH = this adduct as In the present study, we an intramolecular of the linolenate 9-peroxyl radical on lipoxygenase, generating the carbon-centered radical. On the other lipoxygenase can the hydrogen of the doubly allylic at of linolenate the same intermediate carbon-centered radical, = CH−CH = CH−CH = should be However, the of this pathway should be 15-lipoxygenase did not the hydrogen of the doubly allylic at of α-linolenic acid, and of the α-linolenic acid, with a of linolenate for the active on For an intramolecular of the linolenate 9-peroxyl radical, the electron to to the doubly allylic at of the molecule that the between the doubly allylic hydrogen and the oxygen on which the electron is was to be (Fig. This fact indicates that the of the of the linolenate 9-peroxyl radical is not by in the active of In our recent study, we that the binding affinity of the lipid allyl radical to the active on lipoxygenase at lower oxygen content is not that the nitroxyl radical can scavenge the lipid allyl radical (13Koshiishi I. Tsuchida K. Takajo T. Komatsu M. Radical scavenger can scavenge lipid allyl radicals complexed with lipoxygenase at lower oxygen content. Biochem. J. 2006; 395: 303-309Google Scholar). In a the linolenate 9-peroxyl radical is thought to be in the active on lipoxygenase at lower oxygen content, the intramolecular of the linolenate 9-peroxyl radical. The of generation in the reaction solution is shown in Fig. 5 The generation of linolenate hydroperoxide was completed within 1 min. The dioxygenation of a PUFA results in the decreased oxygen content. In our recent study (13Koshiishi I. Tsuchida K. Takajo T. Komatsu M. Radical scavenger can scavenge lipid allyl radicals complexed with lipoxygenase at lower oxygen content. Biochem. J. 2006; 395: 303-309Google Scholar), we that the reaction between the linoleate allyl radical and the nitroxyl radical was not the dioxygenation was shown in Fig. was generated at lower oxygen content. Interestingly, the content within the initial 2 through the This fact indicates that linolenate 9-peroxyl radical is generated in as well as at lower oxygen content. the of an oxygen molecule at the C-13 position of the linolenate allyl radical in is speculated to be by a channel through which the oxygen molecule an electron on the C-13 (7Knapp M.J. Klinman J.P. Kinetic studies of oxygen reactivity in soybean lipoxygenase-1. Biochemistry. 2003; 42: 11466-11475Google Scholar), the oxygen molecule seems to with the carbon-centered radical at the C-9 position of the linolenate allyl radical. Furthermore, this system in is thought to be at lower oxygen content. In fact, the between and was at In our the intramolecular of the linolenate 9-peroxyl radical may on the we to the that fatty acid-derived radicals the of lipoxygenase in the of a conjugated triene through a reaction between a radical and a Furthermore, and Boldingh G.J. Vliegenthart J.F.G. Boldingh J. anaerobic reaction between lipoxygenase, linoleic acid and Biochem. J. Scholar, G.J. Vliegenthart J.F.G. Boldingh J. The and of fatty acids from the anaerobic reaction between lipoxygenase, linoleic acid and Biochem. J. demonstrated that in an anaerobic reaction lipoxygenase, linoleic acid, and the linoleate allyl radical on the lipoxygenase a hydrogen from linoleate generating the linoleate radical. To the of linolenate radical generation from linolenate we the presence of linoleate hydroperoxide the generation of shown in Fig. the linoleate hydroperoxide did not prevent In the of the reaction between the linolenate allyl radical and linolenate hydroperoxide should be an of the nitroxyl radical with linolenate hydroperoxide for the linolenate allyl radical. Remarkably, the of linoleate hydroperoxide in the reaction solution the one-electron redox reaction between α-linolenic acid and linolenate hydroperoxide by lipoxygenase in the presence of It seems that the in hydroperoxides results in the of the in which the ferrous lipoxygenase is converted into the ferric we confirmed that of linolenate hydroperoxide from the α-linolenic acid solution used did not generation in the linolenate/lipoxygenase/CmΔP In we that a lipid radical can be into a hydroperoxy fatty acid allyl radical at lower oxygen content through intramolecular rearrangement. In the present study, we employed α-linolenic acid, this PUFA two doubly allylic at and In arachidonic acid, eicosapentaenoic acid, and acid doubly allylic that the resulting hydroperoxy fatty acid allyl radicals in these intermediate carbon-centered radicals should give rise to of hydroperoxy fatty acids with structural diversity. In a study, we to evaluate the real of the hydroperoxy fatty acids produced in a PUFA/lipoxygenase system at lower oxygen content. 3-carbamoyl-2,2,5,5-tetramethyl-3-pyrroline-N-oxyl liquid chromatography tandem mass spectrometry precursor ion scanning ultraviolet

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