Two Distinct Pathways of Formation of 4-Hydroxynonenal

MECHANISMS OF NONENZYMATIC TRANSFORMATION OF THE 9- AND 13-HYDROPEROXIDES OF LINOLEIC ACID TO 4-HYDROXYALKENALS*

Claus SchneiderDagger , Keri A. Tallman§, Ned A. Porter§, and Alan R. BrashDagger

From the Dagger  Department of Pharmacology, School of Medicine, and § Department of Chemistry, Vanderbilt University, Nashville, Tennessee 37232

Received for publication, February 28, 2001


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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% S configuration, 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.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 (1-3) 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 (4, 5). 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 (6), the oxidative alterations in Alzheimer's disease (7, 8), and its participation in the formation of etheno DNA-base adducts (9).

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 omega -6 essential fatty acids. It was also reported in the early work (4) that 15-hydroperoxy-eicosatetraenoic acid is a precursor. In 1990, Porter and Pryor (10) 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 (11) 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 (11) 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 (12) 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, Noordermeer et al. (13) 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 (14).

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 alpha -tocopherol. The use of chiral starting materials and analyses of stereochemistry of the products reveal a pathway from omega -6 hydroperoxides (13-HPODE) to the 4-hydroxyalkenals and insights into the stereochemistry of the nonenzymatic reactions.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Preparation of Hydroperoxides-- 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 (15) 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.

Autoxidation Reactions-- 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, alpha -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.

Identification of Autoxidation Products-- 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 in Escherichia coli (16). 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 (delta  = 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 (16). After 5 min, the reaction was terminated by adding N HCl up to pH 4.5 and extracted twice with 30 ml of ethyl acetate containing 250 µg alpha -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. The syn- 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 (delta  = 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 (delta  = 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 (17). 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).

Quantification of 4-HPNE and 9-Hydroperxy-12-oxo-10E-dodecenoic acid-- 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.

Chiral Resolution of 4-H(P)NE and 9-Hydro(pero)xy-12-oxo-10E-dodecenoic Acid-- 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 (syn and 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.

CD Spectroscopy-- 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 A239 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.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Time Course of Degradation of Linoleic Acid Hydroperoxides-- 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 alpha -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 alpha -tocopherol the degradation is slowed down to about 70-80% remaining hydroperoxides after 4 h.


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Fig. 1.   Time course of the degradation of linoleic acid hydroperoxides in the presence and absence of alpha -tocopherol. 25-µg aliquots of the hydroperoxides were autoxidized with or without 5% (w/w) alpha -tocopherol (alpha -toc.) at 37 °C and analyzed as described under "Experimental Procedures." The remaining HPODEs are expressed as a percentage of the starting amount (t = 0, set at 100%).

RP-HPLC Analysis of Autoxidation Reactions-- The polar products formed in the autoxidation reactions were analyzed by RP-HPLC (Fig. 2). In these chromatograms, the autoxidations of 13S-HPODE (Fig. 2A) and 9S-HPODE (Fig. 2B) were analyzed after 1 h, and the autoxidation of 13S-HPODE in the presence of 5% (w/w) alpha -tocopherol was analyzed after 4 h (Fig. 2C). 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.


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Fig. 2.   RP-HPLC analysis of the autoxidation reactions of linoleic acid hydroperoxides. A, autoxidation of 13S-HPODE. B, autoxidation of 9S-HPODE. C, autoxidation of 13S-HPODE in the presence of 5% (w/w) alpha -tocopherol (alpha -toc.). 25-µg aliquots of the hydroperoxides were auto-oxidized at 37 °C for 1 h (A and B) or 4 h (C), and the complete reaction mixture was analyzed on RP-HPLC (Waters Symmetry C18 5-µm, 0.46 × 25 cm, 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. The chromatograms shown were recorded at 220 nm (full scale absorbance, 25 milliabsorbance units).

Identification of Products-- Compound 3 was identified as 4-HPNE based on identical UV spectra (lambda max 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.


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Fig. 3.   UV spectra of 4-HNE, compound 3 (4-HPNE), compound 4 (8,13-diHPODE), and 13S-HPODE. The UV spectra were recorded in column solvent (acetonitrile/water/acetic acid 60:40:0.01 by volume) using an HP 1040A diode array detector. The spectra are normalized to lambda max.

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 (16), (iii) autoxidation of the 12-oxo-9Z-dodecenoic acid cleavage product in the presence of alpha -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. 2A) and 6 and 7 (derived from 9S-HPODE, Fig. 2B) 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 a trans,trans conjugated diene (lambda max 231 nm; Fig. 3) (18), 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)3 C5H9OSi(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. 5A). 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: delta  (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 (19). Despite their complexity, the 1H NMR spectra were entirely supportive of the structures deduced from the UV and MS data.


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Fig. 4.   LC-coordination ion spray (Ag+) mass spectrum of 8,13-dihydroperoxy-9E,11E-octadecadienoic acid (compound 5). Compound 5 was isolated from the autoxidation of 5 mg of 13S-HPODE by RP-HPLC and analyzed by LC-CSI-MS with AgBF4 using the conditions described under "Experimental Procedures."


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Fig. 5.   Mass 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.

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,13S configurations. 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. 5B). 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,14R configuration.

Time Course of Formation of 4-HPNE and 9-Hydroperoxy-12-oxo-10E-dodecenoic Acid-- 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. 6A). 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. 6B).


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Fig. 6.   Time course of the formation of 4-HPNE and 9-hydroperoxy-12-oxo-10E-dodecenoic acid from 13S-HPODE (A) and 9S-HPODE (B). 25-µg aliquots of the hydroperoxides were autoxidized at 37 °C for the time indicated and analyzed as described under "Experimental Procedures." 4-HPNE and 9-hydroperoxy-12-oxo-10E-dodecenoic acid were quantified based on external calibration using 4-HNE as a standard.

Autoxidation of 13S-HPODE in the Presence of alpha -Tocopherol-- Autoxidations of 25-µg aliquots of 13S-HPODE as a dry film were carried out in the presence of 5% alpha -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 alpha -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. 2C). 4-HPNE (compound 3) and the UV-absorbing conjugated diHPODEs are only minor products in these experiments.

Chiral Analysis of 4-HPNE-- 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 (Chiralpak AD). Therefore, the aldehyde group was derivatized to the MOX derivative. The resulting syn- and anti-isomers were readily resolved using RP-HPLC. When the later eluting oxime isomer from the RP-HPLC separation was injected on chiral phase HPLC, the enantiomers in a commercial standard of 4-HNE were widely resolved with retention times of 5.7 and 7.4 min (Fig. 7C). The earlier eluting MOX isomer was also resolved into two enantiomers, but with less resolution. The elution order of the enantiomers from the chiral phase HPLC separation was determined using CD spectroscopy (see below).


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Fig. 7.   Chiral phase HPLC analysis of 4-H(P)NE (methoxime derivative) derived from 13S-HPODE (A) and 9S-HPODE (B). 4-HPNE isolated from autoxidations was reduced with triphenylphosphine and derivatized and purified by RP-HPLC as described under "Experimental Procedures." A, analysis of 4-HPNE isolated from the autoxidation of 5 mg of 13S-HPODE. B, analysis of 4-HPNE isolated from the autoxidation of 5 mg of 9S-HPODE. C, racemic reference of 4-HNE. Chiral resolution was performed on a Chiralpak AD column (0.46 × 25 cm) eluted with hexane/ethanol (90:10 by volume) at a flow rate of 1 ml/min and UV detection at 235 nm. The elution order was determined by CD spectroscopy of the collected enantiomers.

From separate 5-mg autoxidations of 13S- and 9S-HPODE, the 4-HPNE product was isolated by RP-HPLC, reduced with triphenylphosphine, and converted to the MOX derivative. The syn- and anti-isomers of the MOX derivative were resolved on RP-HPLC as described above for the racemic standards. Fig. 7A shows the chiral phase HPLC elution profile of the later eluting MOX isomer of 4-HNE derived from 13S-HPODE. Integration of the peak areas of the enantiomers gave a 90:10 ratio of 4S-HNE to 4R-HNE. Similar chiral analysis of the 4-HPNE product from a 9S-HPODE auto-oxidation showed that it was formed as a virtually racemic mixture (54% S and 46% R; Fig. 7B).

Chiral Analysis of 9-Hydroperoxy-12-oxo-10E-dodecenoic Acid-- A method for chiral analysis of 9-hydro(pero)xy-12-oxo-10E-dodecenoic acid was developed for the methyl ester derivative along the same lines as for 4-HNE. Injection of the earlier eluting methyl ester MOX isomer from RP-HPLC resulted in the resolution into two enantiomers as shown in Fig. 8C (retention times of 9.4 and 12.9 min). When the 9-hydroperoxy-12-oxo-10E-dodecenoic acid derived from autoxidation of 5 mg of 13S-HPODE was analyzed using this method, it was found to be an almost racemic mixture (53:47 S to R) (Fig. 8A). In contrast, 9-hydroperoxy-12-oxo-10E-dodecenoic acid was formed from 9S-HPODE in an S/R enantiomer ratio of 91:9 (Fig. 8B).


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Fig. 8.   Chiral phase HPLC analysis of 9-hydroxy-12-oxo-10E-dodecenoic acid methyl ester (methoxime derivative) derived from 13S-HPODE (A) and 9S-HPODE (B). 9-Hydroperoxy-12-oxo-10E-dodecenoic acid isolated from autoxidations was reduced with triphenylphosphine and derivatized and purified by RP-HPLC as described under "Experimental Procedures." A, analysis of 9-hydroperoxy-12-oxo-10E-dodecenoic acid isolated from the autoxidation of 5 mg of 13S-HPODE. B, analysis of 9-hydroperoxy-12-oxo-10E-dodecenoic acid isolated from the autoxidation of 5 mg of 9S-HPODE. C, racemic reference of 9-hydroxy-12-oxo-10E-dodecenoic acid. Chiral resolution was performed on a Chiralpak AD column (0.46 × 25 cm) eluted with hexane/ethanol (90:10 by volume) at a flow rate of 1 ml/min and UV detection at 235 nm. The elution order was determined by CD spectroscopy of the collected enantiomers.

CD Spectroscopy-- The absolute configuration of the enantiomers of the MOX-derivatized 4-HNE and 9-hydroxy-12-oxo-10E-dodecenoic acid was determined by CD spectroscopy to determine the elution order from the chiral phase HPLC separations (20). The enantiomers of both products were collected from chiral phase HPLC, and the hydroxy group was derivatized with 2-naphthoyl-imidazole to introduce a second chromophore at the chiral center as depicted in Fig. 9. The exciton-coupled circular dichroism method of CD spectroscopy uses the interaction of two chromophores at the chiral center to define the absolute configuration. To delineate the absolute configuration of a particular chiral molecule from the CD spectrum, the molecule is represented in the Newman projection. If the chirality of the electric transition moments of the first to the second chromophore is clockwise, defined as positive, the CD shows a positive first and a negative second Cotton effect; if the chirality is counter clockwise, defined as negative, the CD shows a negative first and a positive second Cotton effect.


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Fig. 9.   Derivatization of the 4-HNE methoxime derivative to the 2-naphthoate. The chromophoric derivatives for CD spectroscopy were synthesized by derivatization of the enantiomers of methoxime 4-HNE, collected from the chiral phase separation, with 1-(2-naphthoyl)imidazole. The sign of the Cotton effects of the two enantiomers can be predicted from the left- (-) and right-handed (+) sense between the transition moments (thick black line) of the chromophores as indicated by the curved arrows. In this case, the S enantiomer of the derivatized 4-HNE has positive chirality.

The 2-naphthoate derivative of the first eluting enantiomer of the MOX-derivatized 4-HNE from the chiral column resolution showed a positive first Cotton effect at 244 nm (Delta epsilon , +32.4) and a negative second Cotton effect at 226 nm (Delta epsilon , -27.0) (Fig. 10). Thus, the CD spectrum of this enantiomer has a clockwise (positive) sense between the transition moments of the two chromophores as depicted in the projection in Fig. 9; therefore its absolute configuration is S. The later eluting enantiomer exhibited a mirror-image CD spectrum with extrema at 244 nm (Delta epsilon , -33.1) and 226 nm (Delta epsilon , +27.0), resulting in a negative chirality; the absolute configuration is R (Fig. 10).


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Fig. 10.   CD and UV spectra of R- and S-4-HNE (2-naphthoate, methoxime derivative) in acetonitrile. The enantiomers of the methoxime derivative of 4-HNE were resolved using chiral phase HPLC and further derivatized to the 2-naphthoates (Fig. 9 and "Experimental Procedures"). The CD spectrum of the first eluting enantiomer from the chiral phase separation of methoxime 4-HNE has a positive first and a negative second Cotton effect (solid line); therefore it has positive chirality (S configuration; Fig. 9) (20).


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Scheme 1.  

In the case of 9-hydroxy-12-oxo-10E-dodecenoic acid, the first eluting enantiomer from the chiral column showed a negative split CD curve (extremum at 244 nm, Delta epsilon , -3.4) which defines R configuration for this enantiomer. Accordingly, the second enantiomer had a positive split CD curve (extrema at 244 nm, Delta epsilon , +3.2, and at 225 nm, Delta epsilon , -1.7), which reveals S configuration.

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Analysis of the stereochemistry of 4-hydroxyalkenals formed from chiral hydroperoxides together with the detection of some unusual dihydroperoxides of linoleic acid provide some valuable new insights into the pathways of 4-H(P)NE formation. We found that the 4-HPNE formed from 13S-HPODE largely retains the initial S configuration. This retention of configuration can be explained by the mechanism outlined in Scheme 1. The initial event is the abstraction of an allylic hydrogen at C-8 of 13S-HPODE. This yields a radical that can be localized on C-8, C-10, or C-12. Oxygenation at C-10 forms diastereomeric 10R,S,13S-dihydroperoxides. The formation of these products entails a bis-allylic oxygenation that is precedented in autoxidation (21) and in the soybean lipoxygenase-catalyzed oxygenation of a synthetic substrate, 16,17-dehydro-arachidonic acid (22). The doubly allylic 10-hydroperoxy group is unstable, and cleavage in a Hock rearrangement between C-9 and C-10 yields two aldehyde fragments, 9-oxo-nonanic acid and 4S-HPNE. The Hock rearrangement, which is promoted by protic and Lewis acids, occurs readily for hydroperoxides that have an unsaturated unit attached to the carbon bearing the hydroperoxide group (23, 24). Thus, benzylic, allylic, and dienylic hydroperoxides undergo the rearrangement readily by migration of the unsaturated group from carbon to oxygen while the weak O-O bond fragments. The initial 13S-hydroperoxy group of 13S-HPODE is not directly involved in this reaction sequence, and therefore the 4S-HPNE cleavage product retains its absolute configuration.

The 9-hydroperoxy-12-oxo-10E-dodecenoic acid product (compound 1) from 13S-HPODE is formed through an alternative mechanism (Scheme 2). The first reaction is the Hock cleavage of 13-HPODE between carbons 12 and 13 (24). This yields the two aldehydes hexanal and 12-oxo-9Z-dodecenoic acid. Neither compound was detected in our analyses, because hexanal is volatile and virtually nondetectable in the UV, and 12-oxo-9Z-dodecenoic acid rapidly oxygenates to 9-hydroperoxy-12-oxo-10E-dodecenoic acid (13, 25). The stereochemistry of the 9-hydroperoxy group is predicted to be racemic according to this mechanism (Scheme 2). Chiral analysis of the product from 13S-HPODE (Fig. 8) revealed that it is a 53:47 mixture of the S to the R enantiomer, which is largely in agreement with this mechanism.


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Scheme 2.  

The reactions and products observed upon autoxidation of 9S-HPODE were mechanistically equivalent. In an initial Hock rearrangement, 9S-HPODE is cleaved into 9-oxo-nonanic acid and 3Z-nonenal. 3Z-Nonenal is very rapidly oxygenated to 4-HPNE, which in this case is formed as a racemic mixture (Fig. 7B and Scheme 3A). In plants, the cleavage of the 9S-hydroperoxide is catalyzed by a hydroperoxide lyase (26), and the subsequent nonenzymatic oxygenation of 3Z-nonenal has been described as the source for the formation of 4-HNE in plant tissue (13). There is also, however, an initial allylic hydrogen abstraction at C-14, which is analogous to the H abstraction at C-8 of 13-HPODE. The resulting radical rearranges and can be oxygenated at different positions (i.e. on carbons 10, 12, and 14). Hock cleavage of the 9S,12-dihydroperoxide yields 9S-hydroperoxy-12-oxo-10E-dodecenoic acid and hexanal (Scheme 3B). The predicted S configuration of the 9-hydroperoxy group was confirmed by chiral phase analysis (Fig. 8B).


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Scheme 3.  

An initial hydrogen abstraction at C-8 of 13S-HPODE, as postulated in Scheme 1, predicts the formation of positional isomers of diastereomeric linoleic acid dihydroperoxides. We identified a pair of diastereomeric 8,13-dihydroperoxides, providing evidence of the C-8 hydrogen abstraction. The corresponding 8,13-dihydroxy derivatives of linoleic acid have been characterized previously as minor end products of the heme-catalyzed degradation of 13S-HPODE (27). These dihydroxy derivatives were formed via synthesis of a leukotriene type of allylic epoxide (12,13-epoxy-8,10-octadecadienoic acid) that hydrolyzed to the 8,13-dihydroxides. This mechanism is quite distinct from the route to the 8,13-dihydroperoxides that were the major products under the conditions used in our study. The fact that these 8,13-dihydroperoxides have a trans,trans conjugated diene also provides strong circumstantial evidence for oxygenation at the C-10 position. Such a reaction at C-10 is required to account for the change in configuration of the original 9,10 cis double bond to the trans configuration found in the 8,13-dihydroperoxides (Scheme 4) (28, 29). The change in stereochemistry is allowed by the occurrence of a peroxyl radical at C-10. This permits rotation around the 9,10 bond. The subsequent loss of O2 and formation again of the carbon radical aligns the carbon backbone in the more stable trans,trans configuration. Oxygenation at C-8 and formation of the hydroperoxide gives the stable 8,13-dihydroperoxide diastereomers that we isolated and identified (Scheme 4). The equivalent reactions occurred starting with 9S-HPODE, resulting in characterization of the two 9,14-dihydroperoxides by LC-MS and GC-MS.


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Scheme 4.  

The presence of alpha -tocopherol during the autoxidation of 13S-HPODE caused a slowing in the loss of this substrate and a noticeable change in the pattern of products. As an antioxidant, alpha -tocopherol intercepts peroxyl radicals and in the process forms an alpha -tocopheroxyl radical and the hydroperoxide. This accounts for the slowing of the disappearance of the 13S-HPODE starting material. The tocopheroxyl radical becomes the dominant free radical chain carrier, but it is not a sufficiently strong oxidant to abstract an allylic hydrogen at C-8 of 13-HPODE (30). This results in a selective absence of the dihydroperoxides discussed above and of the 4-HPNE cleavage product. On the other hand, direct Hock cleavage of the 13S-HPODE can still occur, providing 12-oxo-9Z-dodecenoic acid. The tocopheroxyl radical can abstract a doubly allylic hydrogen from C-11 of this intermediate, thus forming racemic 9-hydroperoxy-12-oxo-10E-dodecenoic acid as the major polar product of 13S-HPODE detected in the presence of alpha -tocopherol.

Interesting small deviations from the stereochemistries predicted in the mechanisms in Schemes 1 and 3 point to the existence of additional routes to the 4-hydroxyalkenals. We found that the 4-HPNE formed from 13S-HPODE was significantly less than 100% S in stereochemistry. The hydroperoxide starting materials were >= 98% S configuration, so ~10% of the R enantiomer was formed during the reaction by a pathway that is yet to be elucidated. Initial 9/13 hydroperoxide isomerizations occurring prior to chain cleavage may contribute to the product profiles. It was apparent also that the 4-HPNE formed from the 9S-HPODE was not racemic, but rather it showed a slight preference of the S enantiomer. In this case there must be some transfer of chirality from the 9S starting material to the 4-hydroperoxy product. The same consideration holds true for the formation of the 9-hydroperoxy-12-oxo-10E-dodecenoic acids; they showed similar small deviations from the predicted chiralities.

In conclusion, we provide evidence for at least two independent mechanisms leading from isomeric omega -6 fatty acid hydroperoxides to 4-H(P)NE. Here, we used the two isomeric linoleic acid hydroperoxides as model compounds. Analogous reactions are to be expected with hydroperoxides from other omega -6 fatty acids, especially arachidonic acid. With arachidonic acid, 11- and 15-hydroperoxy-eicosatetraenoic acid are the precursors to form 4-HPNE via the analogous mechanisms. The chiral analysis method of 4-HNE we developed here should be useful in further studies to investigate the mechanism of 4-hydroxyalkenal synthesis. For example, the possibility of an involvement of enzymes such as lipoxygenases or cytochrome P450s could result in biosynthesis of chiral 4-HNE and its analogues. A potential enzyme-initiated pathway to 4-HNE becomes increasingly important as physiological activities of 4-HNE in the sub-micromolar range are uncovered.

    ACKNOWLEDGEMENTS

We thank Markus Voehler for expert help with the NMR analyses and William Boeglin for help with the HPLC analyses.

    FOOTNOTES

* This work was supported by National Institutes of Health Grants GM-53638 and GM-15431.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.

To whom correspondence should be addressed: Dept. of Pharmacology, Vanderbilt University School of Medicine, 23rd Ave. at Pierce, Nashville, TN 37232-6602. Tel.: 615-343-4495; Fax: 615-322-4707; E-mail: alan.brash@mcmail.vanderbilt.edu.

Published, JBC Papers in Press, March 19, 2001, DOI 10.1074/jbc.M101821200

    ABBREVIATIONS

The abbreviations used are: 4-HNE, 4-hydroxy-2E-nonenal; 4-HPNE, 4-hydroperoxy-2E-nonenal; HPODE, hydroperoxy-octadecadienoic acid; 13S-HPODE, 13S-hydroperoxy-9Z,11E-octadecadienoic acid; 9S-HPODE, 9S-hydroperoxy-10E,12Z-octadecadienoic acid; SP, straight phase; HPLC, high pressure liquid chromatography; RP, reverse phase; GC, gas chromatography; MS, mass spectrometry; ESI, electrospray ionization; LC, liquid chromatography; MOX, methoxime; CD, circular dichroism.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

1. Benedetti, A., Comporti, M., and Esterbauer, H. (1980) Biochim. Biophys. Acta 620, 281-296[Medline] [Order article via Infotrieve]
2. Benedetti, A., Esterbauer, H., Ferrali, M., Fulceri, R., and Comporti, M. (1982) Biochim. Biophys. Acta 711, 345-356[Medline] [Order article via Infotrieve]
3. Esterbauer, H., Benedetti, A., Lang, J., Fulceri, R., Fauler, G., and Comporti, M. (1986) Biochim. Biophys. Acta 876, 154-166[Medline] [Order article via Infotrieve]
4. Esterbauer, H., Schaur, R. J., and Zollner, H. (1991) Free Radic. Biol. Med. 11, 81-128[Medline] [Order article via Infotrieve]
5. Comporti, M. (1998) Free Radic. Res. 28, 623-635[Medline] [Order article via Infotrieve]
6. Fazio, V. M., Rinaldi, M., Ciafre, S., Barrera, G., and Farace, M. G. (1993) Mol. Aspects Med. 14, 217-228[Medline] [Order article via Infotrieve]
7. Markesbery, W. R., and Carney, J. M. (1999) Brain Pathol. 9, 133-146[Medline] [Order article via Infotrieve]
8. Keller, J. N., and Mattson, M. P. (1998) Rev. Neurosci. 9, 105-116[Medline] [Order article via Infotrieve]
9. Nair, J., Barbin, A., Velic, I., and Bartsch, H. (1999) Mutat. Res. 424, 59-69[CrossRef][Medline] [Order article via Infotrieve]
10. Porter, N. A., and Pryor, W. A. (1990) Free Radic. Biol. Med. 8, 541-543[Medline] [Order article via Infotrieve]
11. Gardner, H. W., and Hamberg, M. (1993) J. Biol. Chem. 268, 6971-6977[Abstract/Free Full Text]
12. Gardner, H. W., and Grove, M. J. (1998) Plant Physiol. 116, 1359-1366[Abstract/Free Full Text]
13. Noordermeer, M. A., Feussner, I., Kolbe, A., Veldink, G. A., and Vliegenthart, J. F. G. (2000) Biochem. Biophys. Res. Commun. 277, 112-116[CrossRef][Medline] [Order article via Infotrieve]
14. Lee, S. H., and Blair, I. A. (2000) Chem. Res. Toxicol. 13, 698-702[CrossRef][Medline] [Order article via Infotrieve]
15. Matthew, J. A., Chan, H. W.-S., and Galliard, T. (1977) Lipids 12, 324-326[Medline] [Order article via Infotrieve]
16. Tijet, N., Schneider, C., Muller, B. L., and Brash, A. R. (2001) Arch. Biochem. Biophys. 386, 281-289[CrossRef][Medline] [Order article via Infotrieve]
17. Havrilla, C. M., Hachey, D. L., and Porter, N. A. (2000) J. Am. Chem. Soc. 122, 8042-8055[CrossRef]
18. Ingram, C. D., and Brash, A. R. (1988) Lipids 23, 340-344[Medline] [Order article via Infotrieve]
19. Silverstein, R. M., Bassler, G. C., and Morrill, T. C. (1981) Spectrometric Identification of Organic Compounds , 4th Ed. , pp. 181-247, John Wiley & Sons, Inc., New York
20. Schneider, C., Schreier, P., and Humpf, H.-U. (1997) Chirality 9, 563-567[CrossRef]
21. Brash, A. R. (2000) Lipids 35, 947-952[Medline] [Order article via Infotrieve]
22. Corey, E. J., and Nagata, R. (1987) Tetrahedron Lett. 45, 5391-5394
23. Frimer, A. A. (1979) Chem. Rev. 79, 359-387
24. Gardner, H. W., and Plattner, R. D. (1984) Lipids 19, 294-299
25. Gardner, H. W. (1998) Lipids 33, 745-749[Medline] [Order article via Infotrieve]
26. Hatanaka, A. (1993) Phytochemistry 34, 1201-1218[CrossRef]
27. Hamberg, M. (1983) Biochim. Biophys. Acta 752, 191-197[Medline] [Order article via Infotrieve]
28. Porter, N. A., Weber, B. A., Weenen, H., and Khan, J. A. (1980) J. Am. Chem. Soc. 102, 5597-5601
29. Porter, N. A., Caldwell, S. E., and Mills, K. A. (1995) Lipids 30, 277-290[Medline] [Order article via Infotrieve]
30. Bowry, V. W., and Ingold, K. U. (1999) Acc. Chem. Res. 32, 27-34[CrossRef]


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