Photolysis of unsaturated fatty acid hydroperoxides. 3. Products from the aerobic decomposition of methyl 13(S)-hydroperoxy-9(Z),11(E-octadecadienoate dissolved in methanol

In the presence of oxygen, UV-irradiation of a solution of methyl 13(S)-hydroperoxy-9(Z), 11(E)-octadecadienoate (13-HPOD) in methanol yields stereoisomers of methyl 9,13-dihydroxy-10-methoxy-11-(E)-octadecenoate and methyl 9,13-dihydroxy-12-methoxy-10(E)-octadecenoate as major products. The reaction pathway to the products was established by photolysis experiments with labeled 13-HPOD and with intermediates of the reaction route. When methanol was substituted by water in the reaction system, the corresponding trihydroxyoctadecenoic acids were formed. This was confirmed by aerobic photolysis of 13-HPOD (free acid) dissolved in water. Threo and erythro methyl 12, 13-epoxy-11-hydroxy-9(Z)-octadecenoates belong to the minor compounds formed during aerobic photolysis of the 13-HPOD in methanol. Labeling experiments indicated that the threo compound resulted mainly from a rearrangement of the 13-HPOD while the erythro compound is mainly formed via secondary hydroperoxidation.     

Fatty acid hydroperoxides biotransformation by potato tuber cell-free extracts

Cleavage of 13-HPOD, 13-HPOT, 9-HPOD and 9-HPOT by potato tuber cell-free extracts was investigated. 13-HPOD and 13-HPOT enzymes were degraded almost completely while 9-HPOD and 9-HPOT were partially transformed. GC-MS analysis of the volatile compounds formed during the reactions revealed that (Z)-3 hexenal, (E)-2-hexenal, pentenols and dimers of pentene were obtained from 13-HPOT while from 13-HPOD hexanal and pentan-1-ol were formed. No volatile was found when 9-HPO isomers were used as substrate, but colneleic acid was produced. When Triton X-100 was omitted in the extraction buffer, only pentenols and dimers of pentene were identified from 13-HPOT and pentan-1-ol from 13-HPOD. Our results reveal that potato tubers that contain Lox, which forms mainly 9-HPO, are able to metabolise the four HPO isomers. Moreover, 13-HPO cleaving activities are due to two distinct enzymatic systems based on, respectively, homolytic and heterolytic mechanisms. The fact that oxygenation of reaction medium dramatically decreases the amount of product resulting from homolytic cleavage strengthens the hypothesis of an anaerobic reaction due to Lox.     

Hydroperoxide lyases (CYP74C and CYP74B) catalyze the homolytic isomerization of fatty acid hydroperoxides into hemiacetals

The conversion of linoleic acid 9-hydroperoxide (9-HPOD) by recombinant melon (Cucumis melo L.) hydroperoxide lyase (HPL, CYP74C subfamily) was studied. Short (5 s-1 min) incubations at 0 degrees C followed by rapid extraction and trimethylsilylation made it possible to trap a new unstable (t(1/2) <30 s) product, i.e. the hemiacetal (1'E,3'Z)-9-hydroxy-9-(1',3'-nonadienyloxy)-nonanoic acid. Identification was performed by GC-MS analysis and substantiated by the formation of trimethylsilyl 9-trimethylsilyloxy-9-nonyloxy-nonanoate upon catalytic hydrogenation and by (2)H-labelling experiments. Both (18)O atoms of [(18)O(2)-hydroperoxy]9-HPOD were incorporated into the hemiacetal. Along with the hemiacetal, three chain-cleavage products, i.e. the enol (1E,3Z)-nonadienol and the hydrates of 3(Z)-nonenal and 9-oxononanoic acid, were trapped as their trimethylsilyl derivatives. The kinetics of (18)O incorporation from [(18)O(2)]9-HPOD provided strong evidence that the cleavage products originated in the hemiacetal. Linolenic and linoleic acid 13-hydroperoxides served as substrates for recombinant HPLs of melon, alfalfa (Medicago sativa) and guava (Psidium guajava), and in each case hemiacetals and enols were detectable by the trapping technique. The data obtained demonstrated that CYP74C and CYP74B HPLs act as isomerases performing a homolytic rearrangement of fatty acid hydroperoxides into short-lived hemiacetals which upon decomposition produce 3(Z)-nonenal, 3(Z)-hexenal and other short chain aldehydes.     

Peroxidase-like activity of lipoxygenases: different substrate specificity of potato 5-lipoxygenase and soybean 15-lipoxygenase and particular affinity of vitamin E derivatives for the 5-lipoxygenase.

Potato 5-lipoxygenase (5-PLO) catalyzes the reduction of 13(S)-hydroperoxy-9Z,11E-octadecadienoic acid (13-HPOD) in the presence of vitamin E. I mol of vitamin E is required to consume 2 mol of 13-HPOD. The mechanism of the 5-PLO-catalyzed oxidation of vitamin E by 13-HPOD is similar to that previously established for the soybean 15-lipoxygenase (L-1)-catalyzed oxidation of phenidone by 13-HPOD, and seems to involve a one-electron reduction of the O-O bond of 13-HPOD. 5-PLO and L-1 exhibit very different substrate specificities and pH profiles for their peroxidase-like activity. Actually, among the 20 compounds containing various reducible functions and the 10 derivatives of vitamin E which have been studied, only four products containing hydrophobic long chains, ascorbic acid 6-palmitate, the trolox esters of octanol and undecanol, and vitamin E exhibit high peroxidase-like activities for 5-PLO. On the contrary, much more compounds, even not very hydrophobic, are good substrates for the peroxidase-like activity of L-1.     

Free radical oxidation of coriolic acid (13-(S)-hydroxy-9Z,11E-octadecadienoic acid)

The reaction of (13S,9Z,11E)-13-hydroxy-9,11-octadecadienoic acid (1a), one of the major peroxidation products of linoleic acid and an important physiological mediator, with the Fenton reagent (Fe(2+)/EDTA/H(2)O(2)) was investigated. In phosphate buffer, pH 7.4, the reaction proceeded with >80% substrate consumption after 4h to give a defined pattern of products, the major of which were isolated as methyl esters and were subjected to complete spectral characterization. The less polar product was identified as (9Z,11E)-13-oxo-9,11-octadecadienoate (2) methyl ester (40% yield). Based on 2D NMR analysis the other two major products were formulated as (11E)-9,10-epoxy-13-hydroxy-11-octadecenoate (3) methyl ester (15% yield) and (10E)-9-hydroxy-13-oxo-10-octadecenoate (4) methyl ester (10% yield). Mechanistic experiments, including deuterium labeling, were consistent with a free radical oxidation pathway involving as the primary event H-atom abstraction at C-13, as inferred from loss of the original S configuration in the reaction products. Overall, these results provide the first insight into the products formed by oxidation of 1a with the Fenton reagent, and hint at novel formation pathways of the hydroxyepoxide 3 and hydroxyketone 4 of potential (patho)physiological relevance in settings of oxidative stress.     

Thermal conversions of trimethylsilyl peroxides of linoleic and linolenic acids

The trimethylsilyl (TMS) peroxides/esters of the fatty acid hydroperoxides (9S,10E,12Z)-9-hydroperoxy-10,12-octadecadienoic acid (9-HPOD) and (9Z,11E,13S,15Z)-13-hydroperoxy-9,11,15-octadecatrienoic acid (13-HPOT) were subjected to gas chromatography-mass spectrometry and products formed by thermal rearrangements were identified. The main products were decadienals and the TMS derivatives of 13-oxo-9,11-tridecadienoic acid, epoxyalcohols, hemiacetals, and ketodienes. Oxy radicals as well as epoxyallylic radicals served as intermediates in the formation of these compounds. The thermal TMS peroxide conversions documented provided biomimetic models for enzymatic conversions of fatty acid hydroperoxides and also offered a method to generate an array of oxylipin derivatives of value as reference compounds in GC-MS studies.     

Anaerobic lipoxygenase activity from Chlorella pyrenoidosa

The micro-alga Chlorella pyrenoidosa expresses an enzymatic activity that cleaves the 13-hydroperoxide derivatives of linoleic acid [13-hydroperoxy-9(Z),11(E)-octadecadienoic acid, 13-HPOD] and linolenic acid [13-hydroperoxy-9(Z),11(E),15(Z)-octadecatrienoic acid, 13-HPOT] into volatile C(5) and non-volatile C(13) oxo-products. This enzymic activity initially was attributed to a hydroperoxide lyase enzyme; however, subsequent studies showed that this cleavage activity is the result of lipoxygenase activity under anaerobic conditions. Headspace analysis of the volatile products by GC/MS showed the formation of pentane when the substrate was 13-HPOD, whereas a more complex mixture of hydrocarbons was formed when 13-HPOT was the substrate. Analysis of the non-volatile cleavage products from 13-HPOD by liquid chromatography/MS indicated the formation of 13-oxo-9(Z),11(E)-tridecadienoic acid (13-OTA) along with the 13-keto-octadecadienoic acid derivative. When the substrate is 13-HPOT, liquid chromatography/MS analysis indicated the formation of 13-OTA as the major non-volatile product. Aldehyde dehydrogenase (AldDH) oxidizes 13-OTA to an omega-dicarboxylic acid, whereas alcohol dehydrogenase (ADH) reduces 13-OTA to an omega-hydroxy carboxylic acid. AldDH and ADH require the oxidized (NAD(+)) and reduced (NADH) forms of the cofactor NAD, respectively. By combining the action of AldDH and ADH into a continuous cofactor-recycling process, it is possible to simultaneously convert 13-OTA to the corresponding omega-dicarboxylic acid and omega-hydroxy carboxylic acid derivatives.     

Irreversible inactivation of soybean lipoxygenase-1 by hydrophobic thiols

Soybean lipoxygenase-1 is inactivated by micromolar concentrations of the following hydrophobic thiols: 1-octanethiol, 12(S)-mercapto-9(Z)-octadecenoic acid (S-12-HSODE), 12(R)-mercapto-9(Z)-octadecenoic acid (R-12-HSODE), and 12-mercaptooctadecanoic acid (12-HSODA). In each case, inactivation is time-dependent and not reversed by dilution or dialysis. Inactivation requires 13-hydroperoxy-9(Z),11(E)-octadecadienoic acid (13-HPOD), which suggests that it is specific for the ferric form of the enzyme. Lipoxygenase catalyzes an oxygenation reaction on each of the aforementioned thiols, as judged by the consumption of O(2). These reactions also require 13-HPOD. 1-Octanethiol is converted to 1-octanesulfonic acid, which was identified by GC/MS of its methyl ester. The rates of oxygen uptake for R- and S-12-HODE are about 5- and 2.5-fold higher than the rate with 1-octanethiol. The stoichiometries of inactivation imply that inactivation occurs on approximately 1 in 18 turnovers for 12-HSODA, 1 in 48 turnovers for 1-octanethiol, 1 in 63 turnovers for S-12-HSODE, and 1 in 240 turnovers for R-12-HSODE. These data imply that close resemblance to lipoxygenase substrates is not a crucial requirement for either oxidation or inactivation. Under the conditions of our experiments, inactivation was not observed with several more polar thiols: mercaptoethanol, dithiothreitol, L-cysteine, glutathione, N-acetylcysteamine, and captopril. The results imply that hydrophobic thiols irreversibly inactivate soybean lipoxygenase by a mechanism that involves oxidation at sulfur.     

Fatty acid allene oxides. II. Formation of two macrolactones from 12,13(S)-epoxy-9(Z),11-octadecadienoic acid

An unstable fatty acid allene oxide, 12,13(S)-epoxy-9(Z),11-octadecadienoic acid, was recently identified as the product formed from 13(S)-hydroperoxy-9(Z), 11(E)-octadecadienoic acid in the presence of corn (Zea mays L.) hydroperoxide dehydrase (M. Hamberg (1987) Biochim. Biophys. Acta 920, 76-84). The present paper is concerned with the spontaneous decomposition of 12,13(S)-epoxy-9(Z),11-octadecadienoic acid in acetonitrile solution. Two major products were isolated and characterized, i.e. macrolactones 12-keto-9(Z)-octadecen-11-olide and 12-keto-9(Z)-octadecen-13-olide.     

Inhibition of soybean lipoxygenase 1 by N-alkylhydroxylamines

Micromolar concentrations of N-octylhydroxylamine dramatically increase the induction period in the conversion of linoleic acid to 13(S)-hydroperoxy-cis-9,trans-11-octadecadienoic acid (13-HPOD) catalyzed by soybean lipoxygenase 1. The induction period produced by N-octylhydroxylamine is abolished by 13-HPOD but not by the corresponding hydroxy acid. Addition of a catalytic amount of lipoxygenase to a mixture of 13-HPOD and N-octylhydroxylamine results in consumption of approximately 1 mumol of 13-HPOD/mumol of N-octylhydroxylamine present. These results can be explained by a model in which 13-HPOD oxidizes the enzyme from an inactive ferrous form to an active ferric form, as proposed by previous workers, and N-octylhydroxylamine reduces the enzyme back to the ferrous form. Consistent with this model, the ESR signal at g = 6.1 characteristic of ferric lipoxygenase is rapidly abolished by N-octylhydroxylamine and can be regenerated by 13-HPOD. These results provide additional support for earlier proposals that ferric lipoxygenase is the catalytically active form and also establish a novel method of inhibiting enzymes in this class. The octyl group of N-octylhydroxylamine appears to contribute to binding near the iron, since hydroxylamine and N-methylhydroxylamine do not extend the induction period. In the n-RNHOH series, activity passes through an optimum at R = decyl.     

Conversion of 9-D- and 13-L-hydroperoxylinoleic acids by soybean lipoxygenase-1 under anaerobic conditions.

Soybean lipoxygenase-1 reacts with both 9-D and 13-L-hydroperoxylinoleic acids under anaerobic conditions. Approximately 40% of the hydroperoxide is converted into oxodienes, absorbing at 285 nm. Concomitantly, more polar compounds are formed, tentatively identified as being mainly epoxy-hydroxy-octadecenoic acids. When oxygen is present, the reaction is strongly inhibited, until in a very slow reaction the oxygen has been depleted. This accounts for the occurrence of a lag period.     

Carotenoid composition in petals of chrysanthemum (Dendranthema grandiflorum (Ramat.) Kitamura)

Sixteen xanthophylls were isolated from the petals of chrysanthemum (Dendranthema grandiflorum (Ramat.) Kitamura). Among them, (3S,5S,6R,3'R,6'R)-5,6-dihydro-5,6-dihydroxylutein (1) and five di-Z geometrical isomers of lutein-5,6-epoxide, i.e., 9Z,13'Z (2), 13Z,9'Z (3), 9'Z,13'Z (4), 9Z,13Z (5), and 9Z,9'Z (7), had never before been identified as natural products. All of the carotenoids isolated from chrysanthemum, except for (9Z)-violaxanthin, are beta,epsilon-carotene (alpha-carotene) derivatives. The analyses indicate that carotenoids from the petals of chrysanthemum have a very characteristic composition.     

The novel pathway for ketodiene oxylipin biosynthesis in Jerusalem artichoke (Helianthus tuberosus) tubers

The new route of the plant lipoxygenase pathway, directed specifically towards the ketodiene formation, was detected during in vitro experiments with Jerusalem artichoke (Helianthus tuberosus) tubers. Through this pathway (9Z,11E,13S)-13-hydroperoxy-9,11-octadecadienoic acid (13-HPOD) is reduced to corresponding 13-hydroxy acid (13-HOD), which is in turn dehydrogenated into ketodiene (9Z,11E,13S)-13-oxo-9,11-octadecadienoic acid (13-KOD). Dehydrogenation of 13-HOD into 13-KOD was not dependent on the presence of either NAD or NADP, but was strongly dependent on the presence of oxygen. Under anoxic conditions, 13-HOD dehydrogenation was blocked, but addition of 2,6-dichlorophenolindophenol restored it. Sulfite addition fully suppressed the aerobic dehydrogenation of 13-HOD. Hydrogen peroxide is a by-product formed by the enzyme along with 13-KOD. These data suggest that the ketodiene biosynthesis in H. tuberosus tubers is catalyzed by flavin dehydrogenase. (9S,10E,12Z)-9-Hydroxy-10,12-octadecadienoic acid (9-HOD) is dehydrogenated by this enzyme as effectively as 13-HOD, while alpha-ketol, (9Z)-12-oxo-13-hydroxy-9-octadecenoic acid, and ricinoleic acid did not act as substrates for dehydrogenase. The enzyme was soluble and possessed a pH optimum at pH 7.0-9.0. The only 13-HOD dehydrogenase known so far was detected in rat colon. However, unlike the H. tuberosus enzyme, the rat dehydrogenase is NAD-dependent.     

Iron-catalyzed reaction products of alpha-tocopherol with 1-palmitoyl-2-linoleoyl-3-sn-phosphatidylcholine (13S)-hydroperoxide

Alpha-tocopherol was reacted with 1-palmitoyl-2-[(9Z,11E)-(S)-13-hydroperoxy-9,11-octadecadienoyl]-3-sn-phosphatidylcholine (13-PLPC-OOH) in the presence of a lipid-soluble iron chelate, Fe(III) acetylacetonate, in methanol at 37 degrees C. The reaction product was isolated and identified as a mixture of 1-palmitoyl-2-[(10E)-(12S,13S)-9-(8a-dioxy-alpha-tocopherone)-12,13-epoxy-10-octadecenoyl]-3-sn-phosphatidylcholine and 1-palmitoyl-2-[(9Z)-(12S,13S)-11-(8a-dioxy-alpha-tocopherone)-12,13-epoxy-9-octadecenoyl]-3-sn-phosphatidylcholine (TOO-epoxyPLPC), in which the 12,13-epoxyperoxyl radicals derived from 13-PLPC-OOH attacked the 8a-position of the alpha-tocopheroxyl radical. The iron and ascorbate-catalyzed reaction of 13-PLPC-OOH with alpha-tocopherol in phosphatidylcholine (PC) liposomes was assessed by measuring the reaction products of alpha-tocopherol. When 13-PLPC-OOH and alpha-tocopherol were added in saturated dimyristoyl-PC liposomes, the products were TOO-epoxyPLPC, alpha-tocopherylquinone, and epoxy-alpha-tocopherylquinones. In 1-palmitoyl-2-linoleoyl-PC (PLPC) liposomes, alpha-tocopherol could react with both the 13-PLPC-OOH derived 12,13-epoxyperoxyl radicals and the PLPC-derived peroxyl radicals and formed the addition products together with alpha-tocopherylquinone and epoxy-alpha-tocopherylquinones. Therefore, the iron-catalyzed decomposition of phospholipid hydroperoxides primarily produces epoxyperoxyl radicals, which react with the 8a-carbon centered radical of alpha-tocopherol in liposomal systems.     

Purification, stabilization and characterization of tomato fatty acid hydroperoxide lyase

Fatty acid hydroperoxide lyase (HPO-lyase) was purified 300-fold from tomatoes. The enzymatic activity appeared to be very unstable, but addition of Triton X100 and beta-mercaptoethanol to the buffer yielded an active enzyme that could be stored for several months at -80 degrees C. The enzyme was inhibited by desferoxamine mesylate (desferal), 2-methyl-1,2-di-3-pyridyl-1-propanone (metyrapone), nordihydroguaiaretic acid (NDGA), n-propyl gallate and butylated hydroxyanisole, suggesting the involvement of free radicals in the reaction mechanism and the existence of a prosthetic group in the active center. However, no heme group could be demonstrated with the methods commonly used to identify heme groups in proteins. Only 13-hydroperoxides from linoleic acid (13-HPOD) and alpha-linolenic acid (alpha-13-HPOT) were cleaved by the tomato enzyme, with a clear preference for the latter substrate. The pH-optimum was 6.5, and for concentrations lower than 300 microM a typical Michaelis-Menten curve was found with a K(m) of 77 microM. At higher alpha-13-HPOT concentrations inhibition of the enzyme was observed, which could (at least in part) be attributed to 2E-hexenal. A curve of the substrate conversion as a function of the enzyme concentration revealed that 1 nkat of enzyme activity converts 0.7 mumol alpha-13-HPOT before inactivation. Headspace analysis showed that tomato HPO-lyase formed hexanal from 13-HPOD and 3Z-hexenal from alpha-13-HPOT. A trace of the latter compound was isomerized to 2E-hexenal. In addition to the aldehydes, 12-oxo-9Z-dodecenoic acid was found by GC/MS analysis. To a small extent, isomerization to 12-oxo-10E-dodecenoic acid occurred.     

Epoxyalcohol synthase activity of the CYP74B enzymes of higher plants

The CYP74B subfamily of fatty acid hydroperoxide transforming cytochromes P450 includes the most common plant enzymes. All CYP74Bs studied yet except the CYP74B16 (flax divinyl ether synthase, LuDES) and the CYP74B33 (carrot allene oxide synthase, DcAOS) are 13-hydroperoxide lyases (HPLs, synonym: hemiacetal synthases). The results of present work demonstrate that additional products (except the HPL products) of fatty acid hydroperoxides conversion by the recombinant StHPL (CYP74B3, Solanum tuberosum), MsHPL (CYP74B4v1, Medicago sativa), and CsHPL (CYP74B6, Cucumis sativus) are epoxyalcohols. MsHPL, StHPL, and CsHPL converted the 13-hydroperoxides of linoleic (13-HPOD) and α-linolenic acids (13-HPOT) primarily to the chain cleavage products. The minor by-products of 13-HPOD and 13-HPOT conversions by these enzymes were the oxiranyl carbinols, 11-hydroxy-12,13-epoxy-9-octadecenoic and 11-hydroxy-12,13-epoxy-9,15-octadecadienoic acid. At the same time, all enzymes studied converted 9-hydroperoxides into corresponding oxiranyl carbinols with HPL by-products. Thus, the results showed the additional epoxyalcohol synthase activity of studied CYP74B enzymes. The 13-HPOD conversion reliably resulted in smaller yields of the HPL products and bigger yields of the epoxyalcohols compared to the 13-HPOT transformation. Overall, the results show the dualistic HPL/EAS behaviour of studied CYP74B enzymes, depending on hydroperoxide isomerism and unsaturation.     

Epoxyalcohol synthase of Ectocarpus siliculosus. First CYP74-related enzyme of oxylipin biosynthesis in brown algae

Enzymes of CYP74 family play the central role in the biosynthesis of physiologically important oxylipins in land plants. Although a broad diversity of oxylipins is known in the algae, no CYP74s or related enzymes have been detected in brown algae yet. Cloning of the first CYP74-related gene CYP5164B1 of brown alga Ectocarpus siliculosus is reported in present work. The recombinant protein was incubated with several fatty acid hydroperoxides. Linoleic acid 9-hydroperoxide (9-HPOD) was the preferred substrate, while linoleate 13-hydroperoxide (13-HPOD) was less efficient. α-Linolenic acid 9- and 13-hydroperoxides, as well as eicosapentaenoic acid 15-hydroperoxide were inefficient substrates. Both 9-HPOD and 13-HPOD were converted into epoxyalcohols. For instance, 9-HPOD was turned primarily into (9S,10S,11S,12Z)-9,10-epoxy-11-hydroxy-12-octadecenoic acid. Both epoxide and hydroxyl oxygen atoms of the epoxyalcohol were incorporated mostly from [¹⁸O₂]9-HPOD. Thus, the enzyme exhibits the activity of epoxyalcohol synthase (EsEAS). The results show that the EsEAS isomerizes the hydroperoxides into epoxyalcohols via epoxyallylic radical, a common intermediate of different CYP74s and related enzymes. EsEAS can be considered as an archaic prototype of CYP74 family enzymes.     

Formation of free radical metabolites in the reaction between soybean lipoxygenase and its inhibitors. An ESR study

Recent studies showed that soybean lipoxygenase inhibitors like phenidone and nordihydroguaiaretic acid (NDGA) reduce the catalytically active ferric lipoxygenase to its inactive ferrous form. Addition of 13(S)-hydroperoxy-cis-9,trans-11-octadecadienoic acid (13-HPOD) regenerated the active ferric form. In this paper, it is shown that in such a system the inhibitors are oxidized to free-radical metabolites. Incubation of soybean lipoxygenase and linoleic acid with p-aminophenol, catechol, hydroquinone, NDGA, or phenidone resulted in the formation of the one-electron oxidation products of these compounds. Free-radical formation depended upon the presence of the lipoxygenase and 13-HPOD. The free radicals were detected by ESR spectroscopy, and their structure was confirmed by analysis of the spectra, using a computer correlation technique. These data support the proposed mechanism for the inhibition of lipoxygenase by phenolic antioxidants.     

Production of hexanal from hydrolyzed sunflower oil by lipoxygenase and hydroperoxid lyase enzymes

Hexanal was produced from hydrolyzed sunflower oil in two steps: 1) 13-hydroperoxy-9-(Z),11(E)-octadecadienoic acid (13-HPOD) was formed from linoleic acid (100 mM) by soybean lipoxygenase-1 isoenzyme (Lox-1) with O₂, the reaction resulted 68.7 mM 13-HPOD with a yield of 72%. 2) 13-HPOD (15 mM) was cleaved by spinach leaf hydroperoxide lyase resulting 8.2 mM hexanal (54% yield). Hexanal was isolated from the reaction mixture by repeated steam distillation.