Two Distinct Pathways of Formation of 4-Hydroxynonenal

The mechanism of formation of 4-hydroxy-2E-nonenal (4-HNE) has been a matter of debate since it was discovered as a major cytotoxic product of lipid peroxidation in 1980. Recent evidence points to 4-hydroperoxy-2E-nonenal (4-HPNE) as the immediate precursor of 4-HNE (Lee, S. H., and Blair, I. A. (2000)Chem. Res. Toxicol. 13, 698–702; Noordermeer, M. A., Feussner, I., Kolbe, A., Veldink, G. A., and Vliegenthart, J. F. G. (2000) Biochem. Biophys. Res. Commun.277, 112–116), and a pathway via 9-hydroperoxylinoleic acid and 3Z-nonenal is recognized in plant extracts. Using the 9- and 13-hydroperoxides of linoleic acid as starting material, we find that two distinct mechanisms lead to the formation of 4-H(P)NE and the corresponding 4-hydro(pero)xyalkenal that retains the original carboxyl group (9-hydroperoxy-12-oxo-10E-dodecenoic acid). Chiral analysis revealed that 4-HPNE formed from 13S-hydroperoxy-9Z,11E-octadecadienoic acid (13S-HPODE) retains >90% Sconfiguration, whereas it is nearly racemic from 9S-hydroperoxy-10E,12Z-octadecadienoic acid (9S-HPODE). 9-Hydroperoxy-12-oxo-10E-dodecenoic acid is >90%S when derived from 9S-HPODE and almost racemic from 13S-HPODE. Through analysis of intermediates and products, we provide evidence that (i) allylic hydrogen abstraction at C-8 of 13S-HPODE leads to a 10,13-dihydroperoxide that undergoes cleavage between C-9 and C-10 to give 4S-HPNE, whereas direct Hock cleavage of the 13S-HPODE gives 12-oxo-9Z-dodecenoic acid, which oxygenates to racemic 9-hydroperoxy-12-oxo-10E-dodecenoic acid; by contrast, (ii) 9S-HPODE cleaves directly to 3Z-nonenal as a precursor of racemic 4-HPNE, whereas allylic hydrogen abstraction at C-14 and oxygenation to a 9,12-dihydroperoxide leads to chiral 9S-hydroperoxy-12-oxo-10E-dodecenoic acid. Our results distinguish two major pathways to the formation of 4-HNE that should apply also to other fatty acid hydroperoxides. Slight (∼10%) differences in the observed chiralities from those predicted in the above mechanisms suggest the existence of additional routes to the 4-hydroxyalkenals. The mechanism of formation of 4-hydroxy-2E-nonenal (4-HNE) has been a matter of debate since it was discovered as a major cytotoxic product of lipid peroxidation in 1980. Recent evidence points to 4-hydroperoxy-2E-nonenal (4-HPNE) as the immediate precursor of 4-HNE (Lee, S. H., and Blair, I. A. (2000)Chem. Res. Toxicol. 13, 698–702; Noordermeer, M. A., Feussner, I., Kolbe, A., Veldink, G. A., and Vliegenthart, J. F. G. (2000) Biochem. Biophys. Res. Commun.277, 112–116), and a pathway via 9-hydroperoxylinoleic acid and 3Z-nonenal is recognized in plant extracts. Using the 9- and 13-hydroperoxides of linoleic acid as starting material, we find that two distinct mechanisms lead to the formation of 4-H(P)NE and the corresponding 4-hydro(pero)xyalkenal that retains the original carboxyl group (9-hydroperoxy-12-oxo-10E-dodecenoic acid). Chiral analysis revealed that 4-HPNE formed from 13S-hydroperoxy-9Z,11E-octadecadienoic acid (13S-HPODE) retains >90% Sconfiguration, whereas it is nearly racemic from 9S-hydroperoxy-10E,12Z-octadecadienoic acid (9S-HPODE). 9-Hydroperoxy-12-oxo-10E-dodecenoic acid is >90%S when derived from 9S-HPODE and almost racemic from 13S-HPODE. Through analysis of intermediates and products, we provide evidence that (i) allylic hydrogen abstraction at C-8 of 13S-HPODE leads to a 10,13-dihydroperoxide that undergoes cleavage between C-9 and C-10 to give 4S-HPNE, whereas direct Hock cleavage of the 13S-HPODE gives 12-oxo-9Z-dodecenoic acid, which oxygenates to racemic 9-hydroperoxy-12-oxo-10E-dodecenoic acid; by contrast, (ii) 9S-HPODE cleaves directly to 3Z-nonenal as a precursor of racemic 4-HPNE, whereas allylic hydrogen abstraction at C-14 and oxygenation to a 9,12-dihydroperoxide leads to chiral 9S-hydroperoxy-12-oxo-10E-dodecenoic acid. Our results distinguish two major pathways to the formation of 4-HNE that should apply also to other fatty acid hydroperoxides. Slight (∼10%) differences in the observed chiralities from those predicted in the above mechanisms suggest the existence of additional routes to the 4-hydroxyalkenals. Two distinct pathways of formation of 4-hydroxynonenal. Mechanisms of non-enzymatic transformation of the 9- and 13-hydroperoxides of linoleic acid to 4-hydroxyalkenals.Journal of Biological ChemistryVol. 276Issue 34PreviewPage 20833:In Fig. 3, the labels for 4-HNE and 4-HPNE, 3 were reversed. The correct Fig. 3 is shown below. Also, in the associated text on the same page, left column, the first sentence of the paragraph headed "Identification of Products" should read: "Compound 3 was identified as 4-HPNE based on identical UV spectra (λmax 219 nm in RP-HPLC column solvent; Fig.3) and co-chromatography with a synthesized standard." Full-Text PDF Open Access 4-hydroxy-2E-nonenal 4-hydroperoxy-2E-nonenal hydroperoxy-octadecadienoic acid 13S-hydroperoxy-9Z,11E-octadecadienoic acid 9S-hydroperoxy-10E,12Z-octadecadienoic acid straight phase high pressure liquid chromatography reverse phase gas chromatography mass spectrometry electrospray ionization liquid chromatography methoxime circular dichroism The complex processes of lipid peroxidation result in the formation of multiple products with the potential to interact and influence the outcome of normal cellular processes and control mechanisms. The pioneering work of Esterbauer and co-workers (1Benedetti A. Comporti M. Esterbauer H. Biochim. Biophys. Acta. 1980; 620: 281-296Crossref PubMed Scopus (658) Google Scholar, 2Benedetti A. Esterbauer H. Ferrali M. Fulceri R. Comporti M. Biochim. Biophys. Acta. 1982; 711: 345-356Crossref PubMed Scopus (75) Google Scholar, 3Esterbauer H. Benedetti A. Lang J. Fulceri R. Fauler G. Comporti M. Biochim. Biophys. Acta. 1986; 876: 154-166Crossref PubMed Scopus (161) Google Scholar) on the production of cytotoxic molecules in peroxidation reactions led to the discovery of a group of conjugated aldehydes with toxic potential. Within this group, the most abundant member was identified as 4-hydroxy-2E-nonenal (4-HNE)1. In the ensuing years, 4-HNE has achieved status as one of the best recognized and most studied of the cytotoxic products of lipid peroxidation (4Esterbauer H. Schaur R.J. Zollner H. Free Radic. Biol. Med. 1991; 11: 81-128Crossref PubMed Scopus (5936) Google Scholar, 5Comporti M. Free Radic. Res. 1998; 28: 623-635Crossref PubMed Scopus (107) Google Scholar). In addition to studies on its bioactivity, 4-HNE is commonly used as a biomarker for the occurrence and/or the extent of lipid peroxidation. The reviews on the production of 4-HNE include its involvement in cell cycle control (6Fazio V.M. Rinaldi M. Ciafre S. Barrera G. Farace M.G. Mol. Aspects Med. 1993; 14: 217-228Crossref PubMed Scopus (41) Google Scholar), the oxidative alterations in Alzheimer's disease (7Markesbery W.R. Carney J.M. Brain Pathol. 1999; 9: 133-146Crossref PubMed Scopus (745) Google Scholar, 8Keller J.N. Mattson M.P. Rev. Neurosci. 1998; 9: 105-116Crossref PubMed Scopus (220) Google Scholar), and its participation in the formation of etheno DNA-base adducts (9Nair J. Barbin A. Velic I. Bartsch H. Mutat. Res. 1999; 424: 59-69Crossref PubMed Scopus (188) Google Scholar). Despite the volumes of literature on the occurrence and activities of 4-HNE, there are comparatively few studies on how it is formed. It is recognized that linoleic acid and arachidonic acid are among the potential precursors for 4-HNE formation and that the nine carbons of 4-HNE are represented by the last nine carbons of these ω-6 essential fatty acids. It was also reported in the early work (4Esterbauer H. Schaur R.J. Zollner H. Free Radic. Biol. Med. 1991; 11: 81-128Crossref PubMed Scopus (5936) Google Scholar) that 15-hydroperoxy-eicosatetraenoic acid is a precursor. In 1990, Porter and Pryor (10Porter N.A. Pryor W.A. Free Radic. Biol. Med. 1990; 8: 541-543Crossref PubMed Scopus (196) Google Scholar) presented a hypothesis paper that proposed several mechanisms of 4-HNE formation involving the 4,5-epoxy derivative as the intermediate. The first experimental evidence for a pathway from fatty acid hydroperoxides to 4-HNE stemmed from the work of Gardner and Hamberg (11Gardner H.W. Hamberg M. J. Biol. Chem. 1993; 268: 6971-6977Abstract Full Text PDF PubMed Google Scholar) on the biosynthesis of 4-HNE in broad bean extracts. They established that the aldehydic product of the reaction of 9-hydroperoxylinoleic acid with hydroperoxide lyase, namely 3Z-nonenal, can be converted to 4-hydroperoxy-2E-nonenal (4-HPNE) by a reaction of molecular oxygen, mainly catalyzed in this case by a 3Z-alkenal oxygenase. 4-HPNE is a simple reduction step removed from 4-HNE. Gardner and Hamberg (11Gardner H.W. Hamberg M. J. Biol. Chem. 1993; 268: 6971-6977Abstract Full Text PDF PubMed Google Scholar) also substantiated an additional route to 4-HNE via peroxygenase reactions utilizing the co-substrates 3Z-nonenal and 4-HPNE; the existence of a nonenzymatic pathway was also implicated. Subsequent work by Gardner and Grove (12Gardner H.W. Grove M.J. Plant Physiol. 1998; 116: 1359-1366Crossref PubMed Scopus (42) Google Scholar) showed that 3Z-nonenal is a substrate for soybean lipoxygenase, which thus could function as a 3Z-alkenal oxygenase and that the product is 4-HPNE. More recently, Noordermeeret al. (13Noordermeer M.A. Feussner I. Kolbe A. Veldink G.A. Vliegenthart J.F.G. Biochem. Biophys. Res. Commun. 2000; 277: 112-116Crossref PubMed Scopus (30) Google Scholar) implicated nonenzymatic oxygenation of 3Z-alkenals (via 4-hydroperoxy intermediates) as the major pathway of production of 4-HNE and related 4-hydroxyalkenals in plant extracts. 4-HPNE also has been detected as a nonenzymatic breakdown product of 13-HPODE (14Lee S.H. Blair I.A. Chem. Res. Toxicol. 2000; 13: 698-702Crossref PubMed Scopus (231) Google Scholar). In the present work, we utilized both 9-HPODE and 13-HPODE as model fatty acid hydroperoxides to study the mechanisms of nonenzymatic formation of the 4-hydroxyalkenals. Both fatty acid hydroperoxides give rise to 4-H(P)NE, but each has differing rates and susceptibilities to inhibition by α-tocopherol. The use of chiral starting materials and analyses of stereochemistry of the products reveal a pathway from ω-6 hydroperoxides (13-HPODE) to the 4-hydroxyalkenals and insights into the stereochemistry of the nonenzymatic reactions. 13S-HPODE was synthesized from linoleic acid using soybean lipoxygenase (Sigma, Type V) and purified by preparative SP-HPLC (Alltech Econosil silica, 1.0 × 25 cm, hexane/isopropanol/acetic acid 100:1.5:0.1 by volume at 4 ml/min). 9S-HPODE was synthesized using a lipoxygenase preparation from tomato fruit (15Matthew J.A. Chan H.W.-S. Galliard T. Lipids. 1977; 12: 324-326Crossref PubMed Scopus (134) Google Scholar) and purified using the same SP-HPLC conditions as above. The hydroperoxides were stored as a 5 mg/ml stock solution in acetonitrile or methanol under argon at −80 °C. 25-μg aliquots of the linoleic acid hydroperoxides were transferred into 1.5-ml plastic tubes and evaporated from the solvent under a stream of nitrogen. In some experiments, α-tocopherol from a stock solution in ethanol, 5 or 10% (w/w), was added prior to evaporation. The tubes were placed in a 37 °C oven and removed after a 1-, 2-, or 4-h incubation. 30 μl of column solvent was added, and the complete content was injected on an RP-HPLC column (Waters Symmetry C18 5 μm, 0.46 × 25 cm) eluted with a solvent of acetonitrile/water/acetic acid (60:40:0.01 by volume) at a flow rate of 1 ml/min. The column effluent was monitored using an HP 1040A diode array detector. For product identification and chiral analyses, 5-mg aliquots of 13S- and 9S-HPODE were autoxidized for 5 h at 37 °C. 4-HPNE was prepared from an incubation of 1 mg of 9S-HPODE with a crude bacterial lysate of a hydroperoxide lyase from melon fruit expressed inEscherichia coli (16Tijet N. Schneider C. Muller B.L. Brash A.R. Arch. Biochem. Biophys. 2001; 386: 281-289Crossref PubMed Scopus (84) Google Scholar). 4-HPNE (retention time 5.7 min) was isolated using a Waters Symmetry C18 5-μm column (0.46 × 25 cm) eluted with a solvent of acetonitrile/water/acetic acid (60:40:0.01) at a flow rate of 1 ml/min. The collected product was evaporated from acetonitrile, extracted using a 100-mg C18 cartridge (Varian) eluted with diethyl ether, and dried over Na2SO4. An aliquot of the 4-HPNE was reduced with triphenylphosphine to 4-HNE, repurified by RP-HPLC (retention time 4.6 min), and derivatized with bis(trimethylsilyl)trifluoroacetamide to the trimethylsilyl ether derivative. GC-MS analysis yielded the following diagnostic fragments: m/z 199 [M+ − CHO]; m/z 157 [CHO–C2H2–CH–OSi(CH3)3+]; and m/z 129 [CHO–C2H2–CH–OSi(CH3)3+− CO]. 1H NMR spectra of 4-HPNE were recorded in CD3CN on a Bruker WM 400-MHz spectrometer using residual CH3CN as an internal reference (δ = 1.92 ppm): 9.58 ppm, d, J = 7.8 Hz, H1; 6.9 ppm, dd, J= 15.9 Hz, 6.2 Hz, H3; 6.25 ppm, ddd, J = 15.9 Hz, 7.8 Hz, 1.2 Hz, H2; 4.6 ppm, q, J ∼ 6.5 Hz, H4. 9-Hydroperoxy-12-oxo-10E-dodecenoic acid was prepared from a 5-mg reaction of 13S-HPODE with an expressed and purified recombinant hydroperoxide lyase from melon fruit in 25 ml of 50 mm Tris-HCl, pH 7.5 (16Tijet N. Schneider C. Muller B.L. Brash A.R. Arch. Biochem. Biophys. 2001; 386: 281-289Crossref PubMed Scopus (84) Google Scholar). After 5 min, the reaction was terminated by adding 1 n HCl up to pH 4.5 and extracted twice with 30 ml of ethyl acetate containing 250 μg α-tocopherol. The pooled organic phases were washed with water, dried over Na2SO4, and evaporated under reduced pressure. The crude mixture was kept under an atmosphere of oxygen at 37 °C for 7 days. 9-Hydroxy-12-oxo-10E-dodecenoic acid and 9-hydroperoxy-12-oxo-10E-dodecenoic acid were isolated by RP-HPLC using a Beckman Ultrasphere ODS column (1.0 × 25 cm) eluted with acetonitrile/water/acetic acid (37.5:62.5:0.01 by volume) at 4 ml/min (retention times of 5.4 and 7.3 min, respectively). The collected fractions were evaporated from acetonitrile, and the products were extracted using a 100-mg C18 cartridge (Varian) eluted with ethyl acetate and dried over Na2SO4. For the GC-MS analysis, 9-hydroperoxy-12-oxo-10E-dodecenoic acid was reduced with triphenylphosphine, treated with methoxime hydrochloride and ethereal diazomethane, and purified by RP-HPLC. Thesyn- and anti-isomers gave essentially the same fragment ions at m/z 240 [M+− OCH3], 114 [COC2H2CH=NOCH3]+, and 86 [C2H2CH=NOCH3]+.1H NMR spectra were recorded in CDCl3 on a Bruker WM 400 MHz spectrometer using residual CHCl3 as internal reference (δ = 7.26 ppm). 9-Hydroperoxy-12-oxo-10E-dodecenoic acid: 9.61 ppm, d,J = 7.7 Hz, H12; 6.79 ppm, dd, J = 15.9 Hz, 6.3 Hz, H10; 6.30 ppm, ddd, J = 15.9 Hz, 7.7/7.8 Hz, 1.0 Hz, H11; 4.65 ppm, dt, J = 6.2 Hz, 0.9 Hz, H9; 2.36 ppm, t, J = 7.4 Hz, H2. 9-Hydroxy-12-oxo-10E-dodecenoic acid: 9.59 ppm, d,J = 7.8 Hz, H12; 6.82 ppm, dd, J = 15.7 Hz, 4.7 Hz, H10; 6.31 ppm, ddd, J = 15.7 Hz, 7.8 Hz, 1.4 Hz, H11; 4.43 ppm, m, 1H, H9. The 8,13-diHPODEs and 9,14-diHPODEs were isolated from a 5-mg autoxidation of 13S-HPODE or 9S-HPODE, respectively, by RP-HPLC (Beckman Ultrasphere ODS 10 μm, 1.0 × 25 cm eluted with a solvent of acetonitrile/water/acetic acid (50:50:0.01) at a flow rate of 4 ml/min). For 1H NMR analysis, the collected 8,13-diHPODEs were reduced with NaBH4, methylated, and further purified by SP-HPLC using a Whatman Partisil 5-μm column (0.46 × 25 cm) and a solvent of hexane/isopropanol/acetic acid (90:10:0.1 by volume) at a flow rate of 1 ml/min. The 1H NMR spectra were recorded in C6D6 using residual benzene as internal reference (δ = 7.24 ppm). Aliquots of the samples collected from the initial RP-HPLC separation were used for analysis by LC-ESI-MS. LC-coordination ion spray-MS of the Ag+ adduct ions of the linoleic acid dihydroperoxides was performed on a triple-stage quadrupole TSQ7000 instrument (Finnigan, San Jose, CA) using conditions essentially as described (17Havrilla C.M. Hachey D.L. Porter N.A. J. Am. Chem. Soc. 2000; 122: 8042-8055Crossref Scopus (88) Google Scholar). The HPLC parameters were: Beckman Ultrasphere Si column (0.2 × 25 cm) eluted with hexane/isopropanol/acetic acid (90:10:0.1 by volume) at a flow rate of 0.15 ml/min. A solution of AgBF4 in isopropanol (0.3 mm) was mixed to the column effluent before the ESI interface using a syringe pump at a pump rate of 75 μl/min. For the GC-MS analysis the pairs of diastereomers were collected from RP-HPLC, reduced with triphenylphosphine, methylated using ethereal diazomethane, and further purified by SP-HPLC using a Beckman Ultrasphere Si column (0.46 × 25 cm) eluted with hexane/isopropanol/acetic acid (90:10:0.1 by volume) at 1 ml/min. The collected products were hydrogenated using 5% palladium on alumina and treated with bis(trimethylsilyl)trifluoroacetamide/pyridine. GC-MS was performed on a Finnigan Incos 50 mass spectrometer connected to an HP5890A gas chromatograph. For GC, an 8-m OV1701 column was used with a temperature program starting at 150 °C (1 min isotherm) and a rate of 15 °C/min to 300 °C (4 min isotherm). 4-HPNE and 9-hydroperoxy-12-oxo-10E-dodecenoic acid were quantified using an external calibration curve obtained by injecting aliquots of 4-HNE (5–100 ng) on the RP-HPLC system used for product analysis and plotting against peak height. 100 μg of a racemic standard of 4-HNE (Cayman Chemical, Ann Arbor, MI) were reacted with a molar excess of methyl oxime hydrochloride in 20 μl of pyridine at room temperature overnight. The solvent was evaporated, and the residue was dissolved in 1 ml of methylene chloride and washed three times with 500 μl of water to remove residual reagent and pyridine. The 4-HNE methoxime derivatives were separated on a Waters Symmetry C18 5-μm column (0.46 × 25 cm) eluted with a solvent of acetonitrile/water/acetic acid (50:50:0.01 by volume) at a flow rate of 1 ml/min and UV detection at 235 nm. The two isomers (synand anti) eluted at 10.4 and 11.2 min retention time, respectively. The later eluting isomer was analyzed by chiral phase HPLC using a Chiralpak AD (0.46 × 25 cm) column eluted with hexane/ethanol (90:10 by volume) at a flow rate of 1 ml/min and monitored using an HP 1040A diode array detector. 20 μg of the chemically synthesized 9-hydroperoxy-12-oxo-10E-dodecenoic acid were reduced with triphenylphosphine and treated with methyl oxime hydrochloride in pyridine overnight. The sample was extracted, washed, evaporated, and dissolved in 20 μl of methanol. To this solution a few drops of ethereal diazomethane were added, and the sample was evaporated immediately. The syn- and anti-methoxime (MOX) isomers (retention times of 10.0 and 10.8 min, respectively) were isolated from RP-HPLC (Waters Symmetry C18 5-μm column 0.46 × 25 cm) using acetonitrile/water/acetic acid (37.5:62.5:0.01 by volume) as solvent at a flow rate of 1 ml/min. Chiral analysis of the earlier eluting isomer was performed using the chiral phase HPLC conditions described above. From a 5-mg autoxidation of 13S-HPODE and 9S-HPODE (5 h at 37 °C), 4-HPNE and 9-hydroperoxy-12-oxo-10E-dodecenoic acid were isolated by RP-HPLC using a Beckman Ultrasphere ODS 10-μm column (1.0 × 25 cm) eluted with a solvent of acetonitrile/water/acetic acid (50:50:0.01) at a flow rate of 4 ml/min. The products were reduced with an excess of triphenylphosphine and then further derivatized, purified, and analyzed essentially as described for the racemic standards. The enantiomers of the racemic 4-HNE methoxime derivative and methyl-9-hydroxy-12-oxo-10E-dodecenoic acid methoxime derivative were collected from the chiral phase HPLC separations. The four products were evaporated from solvent under a stream of nitrogen and dissolved in 50 μl of dry acetonitrile. 1 μl of 1,8-diazabicyclo[5,4,0]undec-7-ene and a few grains of 1-(2-naphthoyl)imidazole (Fluka) were added. The reaction was kept at room temperature overnight, and the solvent was evaporated. The residue was dissolved in 1 ml of methylene chloride, washed three times with water, and evaporated; finally the naphthoate derivatives were purified by SP-HPLC using a Beckman Ultrasphere Si column (0.46 × 25 cm) eluted with hexane/isopropanol/acetic acid (100:1:0.1 by volume) at a flow rate of 1 ml/min. For UV and CD spectroscopy, the collected products were evaporated from column solvent and dissolved in acetonitrile to a final A 239 nm of 1 absorbance unit (4-HNE derivatives) or 0.2 absorbance units (methyl-9-hydroxy-12-oxo-10E-dodecenoic acid derivatives). CD spectra were recorded on a JASCO J-700 spectropolarimeter. The 13- and 9-linoleic acid hydroperoxides were autoxidized in 25-μg aliquots as a dry film in open 1.5-ml plastic tubes at 37 °C for 1, 2, or 4 h. At each time point column solvent was added to the tubes, and the complete sample was injected on RP-HPLC. The time course of the decay of 9- and 13-HPODE in the presence and absence of α-tocopherol is shown in Fig.1. Over the course of 4 h at 37 °C the plain hydroperoxides are about 90% degraded, whereas in the presence of α-tocopherol the degradation is slowed down to about 70–80% remaining hydroperoxides after 4 h. The polar products formed in the autoxidation reactions were analyzed by RP-HPLC (Fig.2). In these chromatograms, the autoxidations of 13S-HPODE (Fig. 2 A) and 9S-HPODE (Fig. 2 B) were analyzed after 1 h, and the autoxidation of 13S-HPODE in the presence of 5% (w/w) α-tocopherol was analyzed after 4 h (Fig. 2 C). The polar products with distinctive UV chromophores were designated as 1–7. Compounds 1 and 3 were formed from both hydroperoxides. Compounds 2, 4, and 5 were products of the 13-hydroperoxide, and 6 and 7 were products from 9-HPODE. The arrows in Fig. 2 indicate the retention time of 4-HNE, detected only as a minor product from 13S- and 9S-HPODE in these experiments (<5 ng/25 μg HPODE). Over the course of the 4-h period of autoxidation, no additional abundant products were formed, and there were only minor changes in the product pattern. Compound 3 was identified as 4-HPNE based on identical UV spectra (λmax 223 nm in RP-HPLC column solvent; Fig. 3) and co-chromatography with a synthesized standard. Furthermore, treatment of compound 3 with triphenylphosphine yielded a product that co-chromatographed on RP-HPLC with authentic 4-HNE. The UV spectra of compounds 1 and 3 (4-HPNE) were almost indistinguishable, but compound 1 eluted at a much earlier retention time. The reduction of compound 1 with triphenylphosphine resulted in a slightly more polar product on RP-HPLC and with a UV spectrum almost identical to 4-HNE (Fig. 3). Based on the chromatographic and spectroscopic data, compound 1 was suspected to be 9-hydroperoxy-12-oxo-10E-dodecenoic acid, a C-12 aldehyde derivative that retains the original carboxyl group of the starting fatty acid hydroperoxide. An authentic standard of 9-hydroperoxy-12-oxo-10E-dodecenoic acid was synthesized through the following steps: (i) preparation of 13S-HPODE from linoleic acid using soybean lipoxygenase, (ii) cleavage of the hydroperoxide using a recombinant hydroperoxide lyase from melon fruit (16Tijet N. Schneider C. Muller B.L. Brash A.R. Arch. Biochem. Biophys. 2001; 386: 281-289Crossref PubMed Scopus (84) Google Scholar), (iii) autoxidation of the 12-oxo-9Z-dodecenoic acid cleavage product in the presence of α-tocopherol, and (iv) isolation of the 9-hydroperoxy-12-oxo-10E-dodecenoic acid by RP-HPLC. The identification of compound 3 as 9-hydroperoxy-12-oxo-10E-dodecenoic acid was confirmed by 1H NMR and by GC-MS analysis of the triphenylphosphine-reduced methyl ester methoxime derivative. Compound 2 was identified by LC-MS and 1H NMR as 11-oxo-9Z-undecenoic acid (data not shown). This product is not directly involved in the pathways leading to the formation of the 4-hydro(pero)xyalkenals. Further characterization of this product and its mechanism of formation will be reported elsewhere. As determined by the RP-HPLC analysis, compounds 4 and 5 (derived from 13S-HPODE; Fig. 2 A) and 6 and 7 (derived from 9S-HPODE, Fig. 2 B) were formed consistently in the same relative amount to each other during the 4-h time period of autoxidation. They showed identical UV spectra indicative of atrans,trans conjugated diene (λmax231 nm; Fig. 3) (18Ingram C.D. Brash A.R. Lipids. 1988; 23: 340-344Crossref PubMed Scopus (44) Google Scholar), giving the strong implication that these products were pairs of diastereomers. LC-ESI-MS analysis of the Ag+-adduct ion revealed two [M + Ag+] adduct ions at m/z 451 and 453 for compounds 4, 5, 6, and 7 (Fig. 4). This corresponds to a molecular weight of 344, which is compatible with linoleic acid dihydroperoxides. To reveal the position of the hydroperoxide groups on the fatty acid carbon chain, GC-MS analysis (electron impact mode) was performed on the reduced, methylated, and hydrogenated bis-trimethylsilyl-ether derivatives. The mass spectra of derivatized compounds 4 and 5 (derived from 13S-HPODE) showed characteristic ions at m/z 459 [M − CH3]+, 245 [CH3CO2 C8H13OSi(CH3)3]+and 331 [HCOSi(CH3)3C5H9OSi(CH3)3 C5H11]+(indicating the C-8 hydroxyl), and 403 [CH3CO2C8 H13OSi(CH3)3C5H9OSi(CH3)3]+and 173 [HCOSi(CH3)3 C5H11]+(indicating the C-13 hydroxyl) (Fig.5 A). Finally, 1H NMR of the reduced compounds 4 and 5 (8,13-dihydroxyoctadecadienoates) fully supported the structures. 1H NMR (400 MHz, in deuterated benzene using 7.24 ppm for the residual protons in the solvent) gave for the methyl ester of the 8,13-dihydroxyoctadecadienoate derivative of compound 4: δ (ppm) 0.94, t, 3 protons, H18; 0.98 (d, two protons, –OH, J8,-OH = J13,-OH = 3.5 Hz); 1.2–1.8 (m, 18 protons, H3-H7, and H14-H17); 2.14 (t, 2 protons, H2); 4.00 (m, 2 protons, H8, H13); 5.67 (m, 2 protons, H9, H12); and 6.24 (m, 2 protons, H10, H11). These data confirm the symmetry of the 1,6-dihydroxy-2,4-diene system; the olefinic region shows two complex multiplets, each comprised of two superimposed protons (H10/H11 at 6.24 ppm and H9/H10 at 5.67 ppm), and similarly a superimposed signal for the two geminal hydroxy protons (H8, H13 at 4.00 ppm). The fact that these signals consist of overlapping pairs of protons of identical chemical shift results in nonlinear effects that precluded a ready assignment of the coupling constant across the double bonds. For example, decoupling of the signal for H9/H12 at 5.67 ppm caused the downfield signal for the internal pair of olefinic protons to simplify to a singlet (rather than a doublet), a predictable result based on the lack of coupling between the two superimposed proton signals for H10/H11 (19Silverstein R.M. Bassler G.C. Morrill T.C. Spectrometric Identification of Organic Compounds. 4th Ed. John Wiley & Sons, Inc., New York1981: 181-247Google Scholar). Despite their complexity, the 1H NMR spectra were entirely supportive of the structures deduced from the UV and MS data.Figure 5Mass spectra (electron impact mode) of 8,13-dihydroperoxy-9E,11E-octadecadienoic acid (A) and 9,14-dihydroperoxy-10E,12E-octadecadienoic acid (B) of the reduced, methylated, and hydrogenated trimethylsilyl ether derivative. A, mass spectrum of compound 5 isolated from the autoxidation of 13S-HPODE after reduction, hydrogenation, and derivatization with diazomethane and bis(trimethylsilyl)trifluoroacetamide. B, mass spectrum of compound 7 isolated from the autoxidation of 9S-HPODE after similar derivatization.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Thus, analysis by LC-MS, GC-MS, UV spectroscopy, and 1H NMR identified compounds 4 and 5 as 8,13-dihydroperoxyoctadeca-9E,11E-dienoic acids, presumably a pair of diastereomers with the 8R,13S and 8S,13Sconfigurations. GC-MS analysis of the equivalent derivatives of compounds 6 and 7 derived from 9S-HPODE showed major fragments at m/z 459 [M − CH3]+, 259 [CH3CO2C9H15OSi(CH3)3]+and 317 [HCOSi(CH3)3C5H9OSi(CH3)3C4H9]+(indicating the C-9 hydroxyl), and 417 [CH3CO2C9H15OSi(CH3)3C5H9OSi(CH3)3]+and 159 [HCOSi(CH3)3C4H9]+(indicating the C-14 hydroxyl) (Fig. 5 B). Compounds 6 and 7 were thus identified as 9,14-dihydroperoxyoctadeca-10E,12E-dienoic acids, also presumably a pair of diastereomers with the 9S,14S and 9S,14Rconfiguration. In Fig.6 the time course of the formation of 4-HPNE (compound 3) and 9-hydroperoxy-12-oxo-10E-dodecenoic acid (compound 1) during the autoxidation of 13S- and 9S-HPODE is shown. Starting with 25 μg of 13S-HPODE, ∼100 ng of 4-HPNE are detected after 4 h of autoxidation, whereas from the same amount of 9S-HPODE, less than half as much 4-HPNE is detected (Fig. 6 A). In the formation of 9-hydroperoxy-12-oxo-10E-dodecenoic acid, more is generated from 9S-HPODE (∼100 ng from 25 μg) than from 13S-HPODE (∼30 ng from 25 μg) (Fig.6 B). Autoxidations of 25-μg aliquots of 13S-HPODE as a dry film were carried out in the presence of 5% α-tocopherol for 1, 2, and 4 h. As shown in Fig. 1, the rate of decay of the hydroperoxide is decreased in the presence of α-tocopherol. A representative chromatogram obtained after 4-h autoxidation at 37 °C shows the formation of compound 1 (9-hydroperoxy-12-oxo-10E-dodecenoic acid) as the major product with absorbance at 220 nm (Fig. 2 C). 4-HPNE (compound 3) and the UV-absorbing conjugated diHPODEs are only minor products in these experiments. To provide insights into the mechanism(s) of formation of 4-HPNE, an HPLC method for the chiral resolution of the more stable reduction product 4-HNE was developed. Injection of underivatized 4-HNE on the chiral column used resulted in the reaction of 4-HNE with the chiral stationary phase (Chiralp