Rat testis lipoxygenase-like enzyme. Characterization of products from linoleic acid.

The linoleate oxidation products of the affinity chromatography-purified lipoxygenase-like enzyme isolated from rat testes microsomes were characterized. Three types of reaction products separated by thin-layer chromatography were generally present: polar byproducts (A and B) and hydroperoxides. The methyl hydroxystearates obtained from the enzymically produced hydroperoxides were analysed by gas-liquid chromatography and showed a ratio of 67% 13-hydroxy isomer to 33% 9-hydroxy isomer. The major polar byproduct was analysed by infrared spectra, nuclear magnetic resonance and mass spectrometry (of the toluene-p-sulphonyl derivative) and its structure was established as 13-hydroxy-12-oxo-octadec-cis-9-enoic acid. The possibility of the existence of a linoleate hydroperoxide isomerase in the affinity-purified preparation is discussed.     

Lipoxygenase-like enzyme in rat testis microsomes.

Microsomes, separated from rat testes, were found capable of oxidizing linoleate and arachidonate. The enzyme activity was solubilized with 1% Triton X-100 in acetate buffer (pH 5.0) and purified by affinity chromatography. The overall purification from the starting preparation was approx. 40-fold. The affinity-purified enzyme was almost homogeneous as determined by electrophoresis in polyacrylamide gel. The enzyme was characterized as lipoxygenase-like from its spectrum, specificity, effect of linoleate on its fluorescence and linoleate oxidation products. Three types of compounds separated by thin-layer chromatography were generally present in the lipoxygenase-like enzyme reaction on linoleic acid: substrate fatty acid, polar by-products and hydroperoxides. The hydroperoxides were analyzed by infrared spectra and mass spectrometry and showed the presence of both 9- and 13-hydroxy isomers.     

Hydroperoxide isomers and ketohydroxy product from oxidation of linoleic acid by eggplant lipoxygenase

Hydroperoxides produced by oxidation of linoleic acid with purified eggplant lipoxygenase were separated by TLC and analysed by IR spectroscopy. The methyl hydroxystearates from the enzymatically produced hydroperoxides were analysed by MS and GLC. Both analyses indicated that the eggplant enzyme converted linoleic acid almost exclusively (96%) into the 13-hydroperoxy isomer whereas the 9-hydroperoxy isomer was only a minor product (4%). HPLC of the methyl ester of the isolated hydroperoxides showed three components. Each component was collected, reduced to methyl hydroxystearate and characterized by GLC, MS and IR analysis. The components were identified as 13-hydroperoxy cis-trans isomer (92.8%), 13-hydroperoxy trans-trans isomer (2.6%) and 9-hydroperoxy cis-trans isomer (4.6%). A polar by-product present in the reaction mixture was identified by IR, ¹H NMR, and MS (of the toluene-p-sulphonyl derivative) as 13-hydroxy-12-oxo-octadec-cis-9-enoic acid.     

Decomposition products of Dimers Arising from Secondary Oxidation of Methyl Linoleate Hydroperoxides

Dimers formed in aerated methyl linoleate hydroperoxides were decomposed in liquid paraffin by bubbling with dry air at 30°C for 24 hr to identify the decomposition products. The aerated dimers were fractionated according to their molecular weights by gel permeation chromatography. Identification of the monomeric (25.6%) and low molecular fission products (10.8%) by gas chromatography-mass spectrometry showed the major monomers as methyl hydroxy-octadecadienoate, methyl hydroxy (or hydroperoxy)-epoxy-octadecenoate, methyl dihydroxy (or hydroperoxy)-octadecenoate, methyl trihydroxy (or hydroperoxy)-octadecenoate; and the major fission products as methyl 8-hydroxy-octanoate, 4-hydroxy (or hydroperoxy)-nonanal or -2-nonenal, methyl 12-oxo-9-hydroxy (or hydroperoxy)-dodecanoate or -10-dodecenoate, and methyl 11-oxo-9-undecenoate.

The monomeric products were presumed to be derived from alkoxy radicals generated by the cleavage of peroxy linkages in the dimers, whereas the low molecular products were suggested to be raised by the direct carbon-carbon scission of oxygenated ester moieties on both sides of the peroxy bonds.     

Cationic substrates of soybean lipoxygenase-1

Soybean lipoxygenase-1 (SBLO-1) catalyzes the oxygenation of 1,4-dienes to produce conjugated diene hydroperoxides. The best substrates are anions of fatty acids; for example, linoleate is converted to 13(S)-hydroperoxy-9(Z),11(E)-octadecadienoate. The manner in which SBLO-1 binds substrates is uncertain. In the present work, it was found that SBLO-1 will oxygenate linoleyltrimethylammonium ion (LTMA) to give primarily13(S)-hydroperoxy-9(Z),11(E)-octadecadienyltrimethylammonium ion. The rate of this process is about the same at pH 7 and pH 9 and is about 30% of the rate observed with linoleate at pH 9. At pH 7, SBLO-1 oxygenates linoleyldimethylamine (LDMA) to give primarily 13(S)-hydroperoxy-9(Z),11(E)-octadecadienyldimethylamine. The oxygenation of LDMA occurs at about the same rate as LTMA at pH 7, but more slowly at pH 9. The results demonstrate that SBLO-1 will readily oxygenate substrates in which the carboxylate of linoleate is replaced with a cationic group, and the products of these reactions have the same stereo- and regiochemistry as the products obtained from fatty acid substrates.     

Lipoxygenase-mediated cleavage of fatty acids to carbonyl fragments in tomato fruits

Homogenates of tomato fruits catalysed the enzymic conversion of linoleic and linolenic acids (but not oleic acid) to C₆ aldehydes in low (3–5%) molar yield. Hexanal was formed from linoleic acid; cis-3-hexenal and smaller amounts of trans-2-hexenal were formed from linolenic acid. With the fatty acids as substrates, the major products were fatty acid hydroperoxides (50–80% yield) and the ratio of 9- to 13-hydroperoxides as isolated from an incubation with linoleic acid was at least 95:5 in favour of the 9-hydroperoxide isomer. When the 9- and 13-hydroperoxides of linoleic acid were used as substrates with tomato homogenates, the 13-hydroperoxide was readily cleaved to hexanal in high molar yield (60%) but the 9-hydroperoxide isomer was not converted to cleavage products. Properties of the hydroperoxide cleavage system are described. The results indicate that the C₆ aldehydes are formed from C₁₈ polyunsaturated fatty acids in a sequential enzyme system involving lipoxygenase (which preferentially oxygenates at the 9-position) followed by a hydroperoxide cleavage system which is, however, specific for the 13-hydroperoxy isomers.     

Effect of 1-n-Decylimidazole on Gibberellin Biosynthesis in Tan-ginbozu,a Dwarf Variety of Rice

Previous structural studies of less-polar dimers in autoxidized methyl linoleate (ML) have been extended to polar dimers. After isolation by successive silicic acid and gel permeation chromatography, the dimeric fraction of linoleate was separated into two major fractions, A₁ and A₂, according to their polarities. The polar dimers (A₁) were further fractionated by HPLC either directly or after reduction with triphenyl phosphine on a micro silica column. Isolated subfractions were characterized by UV, IR, GC-MS and FD-MS after suitable derivatizations. FD-MS of all these dimers showed a molecular ion peak which corresponds to 2 × ML + 6 × O and the reduction of each subfraction with stannous chloride gave equimolar amounts of 9 and 13-hydroxy octadecadienoate, and 9, 10, 13 and/or 9, 12, 13-trihydroxy octadecenoate. These results combined with others show that the A₁ dimers are composed of isomeric mixtures containing a peroxide bridge linking a methyl octadecadienoate and a 9, 12 and/or 10, 13-dihydroperoxy octadecenoate across C-9 and/or 13 on each of them.     

Presence of regio-isomeric cholesteryl linoleate hydroperoxides and hydroxides in plasma from healthy humans as evidence of free radical-mediated lipid peroxidation in vivo

Abstract

Lipid hydroperoxides are the primary stable products of lipid peroxidation. We have developed an ultrasensitive method for the detection of lipid hydroperoxides¹ and found about 3 nM cholesteryl ester hydroperoxides (CE-OOH), mostly cholesteryl linoleate hydroperoxides (Ch18:2-OOH), in blood plasma obtained from healthy subjects.² Autoxidation of cholesteryl linoleate (Ch18:2) gives cholesteryl 13-hydroperoxy-9Z,11E-octadecadienoate (13ZE-Ch18:-OOH), cholesteryl 13-hydroperoxy-9E,11E-octadecadienoate (13EE-Ch18:2-OOH), cholesteryl 9-hydroperoxy-10E,12Z-octadecadienoate (9EZ-Ch18:2-OOH), and cholesteryl 9-hydroperoxy-10E,12E-octadecadienoate (9EE-Ch18:2-OOH). Enzymatic oxidation of Ch18:2 with 15-lipoxygenase gives predominantly only one product (13ZE-Ch18:2-OOH).³ To help elucidate the production mechanisms of cholesteryl linoleate hydroperoxides in vivo, we examined the distribution of Ch18:2-O(O)H regioisomers in human blood plasma.     

Oxidation of [1-C]linoleic acid in isolated microsomes from pea leaves

The oxidation of [1-¹⁴C]linoleate in isolated microsomes from pea leaves was found to be stimulated by NADPH addition. The formation of one of the main metabolites, 12-hydroxy-9(Z)-dodecenoic acid is particularly NADPH-dependent. The predominant products in the absence of NADPH were hydroperoxides and in the presence of NADPH, 12-hydroxy-9(Z)-dodecenoic acid. Exogenous [1-¹⁴C]-13-hydroperoxy-9(Z), 11(E)-octadecadieoic acid and [1-¹⁴C]-12-oxo-9(Z)-dodecenoic acidwere the efficient precursors of 12-hydroxy9(Z)-dodecenoic acid. It was concluded that 12-hydroxy-9(Z)-dodecenoic acid is formed by NADPH-dependent enzymatic reduction of 12oxo-9(Z)-dodecenoic acid. The observed inhibition of linoleate oxidation in isolated microsomes by CO and metryapone suggests the involvement of cytochrome P-450 in the reaction. The relative contribution of lipoxygenase and monooxygenase activity to linoleate oxidation in microsomes is discussed.     

Characterization of fluorescent products from reaction of methyl linoleate hydroperoxides with adenine in the presence of Fe2+ and ascorbic acid

The structures of fluorescent products formed in the reaction of methyl linoleate hydroperoxides with adenine, FeSO4 and ascorbic acid were investigated to elucidate the mechanism of interaction. The fluorescent products consisted of at least four major components (I-IV), which could be separated by thin-layer chromatography and high-performance liquid chromatography. Both 2-octenal and 2,4-decadienal, degradation products of methyl linoleate hydroperoxides, reacted with adenine to produce a fluorescent product similar to one of the major compounds (II) formed in the reaction of methyl linoleate hydroperoxides. Spectroscopic data suggest that I and III are the same type of compounds, which have closed ring structures with alpha, beta-unsaturated carbonyl groups between the amino group at the 6-position and the nitrogen at the 1-position of adenine. Component II has a closed ring structure at the same site as I and III, and the presence of an ether linkage was suggested. On the basis of these structures, the involvement of 3-nonenal, methyl 12-oxo-9-dodecenoate and 2-octenal was suggested in the interaction of the methyl linoleate hydroperoxides decomposition products and adenine or DNA in the presence of FeSO4 and ascorbic acid.     

Crystallization and positional specificity of hydroperoxidation of Fusarium lipoxygenase.

A lipoxygenase obtained from the fungus Fusarium oxysporum was purified and crystallized. Using the purified enzyme, the positional specificity of linoleate peroxidation was studied. Linoleate hydroperoxides were converted into the corresponding trimethylsilyl derivative by reduction, catalytic hydrogenation and treatment with hexamethyldisilazane/trimethylchlorosilane/pyridine and then analyzed by combined gas-liquid chromatography-mass spectrometry. Fusarium lipoxygenase was found to produce 9- or 13-hydroperoxy-octadecadienoates from linoleate. The ratio of 9- to 13-hydroperoxides produced by the enzyme was also determined by high performance liquid chromatography of their methyl esters. When the enzymic reaction proceeded at pH 9.0 and 12.0, the ratio of 9- to 13-hydroperoxide isomers was 70 : 30 and 56 : 44, respectively. With the use of the heavy isotope of oxygen (18O2), atoms of oxygen introduced into hydroperoxides were found to be derived from the gaseous phase and not from the aqueous phase.     

Regioisomeric distribution of cholesteryl linoleate hydroperoxides and hydroxides in plasma from healthy humans provides evidence for free radical-mediated lipid peroxidation in vivo

We have previously reported the detection of cholesteryl ester hydroperoxides, consisting mainly of cholesteryl linoleate hydroperoxides (Ch18:2-OOH), at nm levels in plasma from healthy humans (Y. Yamamoto and E. Niki, 1989. Biochem. Biophys. Res. Commun. 165: 988-993). To elucidate their production mechanism in vivo, we examined the distribution of Ch18:2-O(O)H regioisomers in blood plasma from nine healthy young subjects using a sequential method consisting of methanol/hexane extraction in the presence of antioxidant, reductant, and internal standard, solid phase extraction to remove unoxidized cholesteryl linoleate, purification by reversed-phase high-performance liquid chromatography (HPLC), and detection by normal phase HPLC. Furthermore, we confirm that little artifactual oxidation of cholesteryl linoleate occurred during analytical procedures indicated by the absence of oxidation products of cholesteryl 11Z,14Z-eicosadienoate (Ch20:2) when provided as an exogenous substrate to the experimental procedure. We detected nm levels of all free radical-mediated oxidation products, 13ZE-, 13EE-, 9-EZ-, and 9-EE-forms of Ch18:2-O(O)H, in blood plasma, whereas the 13ZE-isomer resulting from enzymatic 15-lipoxygenase oxidation was not evident as a major product. These results indicate that free radical chain oxidation of lipids occurs even in healthy young individuals.     

Further Oxygenated Compounds Produced from Methyl Linoleate Monohydroperoxides at the Process of Autoxidation

The primary stable products, methyl linoleate monohydroperoxides (MLHPO), formed by the autoxidation of methyl linoleate were characterized by gas chromatography-mass spectrometry. MLHPO was converted into methyl hydroxy stearates which consisted of two isomers, methyl 9-hydroxy and methyl 13-hydroxy stearate. Trimethylsilyl ether derivatives of these hydroxy isomers were separated directly by gas chromatography and mass fragmentgraphy. MLHPO was degradated by incubating under aerobic condition at 37°C for a week, and the quantity of MLHPO was determined as hydroxy derivatives. Decrease of MLHPO was almost similar to that of conjugated diene structure, but the peroxide value was not appreciably decreased during the incubation. This fact based on the formation of further oxygenated compounds. After chemical reduction, these compounds were identified as methyl 9,13-hydroxy octadecenoate and methyl 9,12,13- or 9,10,13-trihydroxy octadecenoate, in which oxygen attached to the conjugated diene. The formation mechanisms of these oxygenated compounds are proposed.     

Structures of Dimers Produced from Methyl Linoleate during Initial Stage of Autoxidation

A structural investigation on the main fraction of dimers formed during the induction period of autoxidation in methyl linoleate (ML) was carried out. The dimeric fraction (A₂), which was isolated from the autoxidized ML (POV= 18) by various Chromatographic techniques and gave a single spot on TLC, was further separated into four major components (components 1–4) by high performance liquid chromatography (HPLC). The mean molecular weights of these components were found to be 643–655 and component 4 gave the parent peak 652 on an FD-mass spectrum which corresponded to 2 × ML + 4O. The reduced products of each component with stannous chloride were identified in common as methyl 9- or 13-hydroxy octadecadienoate and methyl 9,13-dihydroxy octadecenoate by GC-MS. These results show that all of these dimers contained a peroxide bridge linking between a pair of MLs across C-9 or C-13 on one of the MLs and C-9 or C-13 on the other, with a hydroperoxy group.     

Flavour biogenesis. Partial purification and properties of a fatty acid hydroperoxide cleaving enzyme from fruits of cucumber

A membrane-bound enzyme, which catalyses the cleavage of fatty acid hydroperoxides to carbonyl fragments, has been partially purified from cucumber fruit. The isomeric 9- and 13-hydroperoxydienes (but not the hydroxydienes) derived from both linoleic and linolenic acids are cleaved by the enzyme but a mixture of 9- and 10-hydroperoxymonoenoic derivatives of oleic acid was not attacked. No evidence was obtained for free intermediates between fatty acid hydroperoxides and the cleavage products. Major volatile products were: cis-3-nonenal and hexanal (from 9- and 13-hydroperoxides of linoleic acid respectively) or cis-3,cis-6-nonadienal and cis-3-hexenal (from 9- and 13-hydroperoxides of linolenic acid). The increase in the ratio of cis-3- to trans-2-enal products with enzyme purification indicated that cis-3-enals are the immediate cleavage products and that the trans-2- forms are produced by subsequent isomerization.     

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.     

Gas Chromatography Mass Spectrometric Analysis of Monohydro-peroxide Isomers and Their Secondary Oxidation Productsof Methyl Linoleate and Methyl Linolenate

Emulsions of methyl linoleate monohydroperoxides (18:2-monoHP) and methyl linolenate monohydroperoxides (18: 3-monoHP) were incubated with ferrous sulfate and ascorbic acid. Gas chromatography mass spectrometric analysis of the trimethylsilyl and te^butyldimethylsilyl derivatives of the reaction products showed that isomerization and secondary oxidation happen competitively during decomposition of 18:2-monoHP, while the secondary oxidation reaction proceeds preferentially and little isomerization is observed in 18: 3-monoHP. It is suggested that 18:3-monoHP is more susceptible to secondary oxidation than 18:2-monoHP because of 18:3 specific secondary oxidation resulting in hydroperoxy-cyclic peroxides and dihydroperoxides. Moreover, an experiment using ¹⁸0₂ has demonstrated that molecular oxygen is scrambled by isomerization and secondary oxidation. It was confirmed that molecular oxygen is attached preferentially to the C-13 position in the 9-monoHP isomer and C-9 position in the 13-monoHP isomer during degradation of 18:2-monoHP.     

Preferential formation of the hydroperoxide of linoleic acid in choline glycerophospholipids in human erythrocytes membrane during peroxidation with an azo initiator

The formation of phospholipid hydroperoxides was monitored in human red blood cell (RBC) membranes that had been peroxidized with an azo initiator. Peroxidation of RBC membranes caused a profound decrease in the amount of polyunsaturated fatty acids and concomitantly hydroperoxides, as primary products of peroxidation, appeared in the phospholipids. Hydroperoxides were predominantly generated in choline glycerophospholipid (CGP), while the extent of formation of ethanolamine glycerophospholipid (EGP) hydroperoxides was low and their presence was transient. Hydroxy and hydroperoxy moieties in CGP were identified as 9-hydroxy and 13-hydroxy octadecanoic acid, derived from linoleic acid, by gas chromatography-mass spectrometric analysis. No consistent generation of hydroperoxide from arachidonic acid was evident in CGP. The CGP-hydroperoxide accounted for approximately 76% of linoleic acid consumed during peroxidation of RBC membranes. The prominent generation of phospholipid hydroperoxides was observed in the linoleic acid-rich membranes from rabbit RBC, indicating that the level of linoleic acid in phospholipids determins, in part, the extent of formation of phospholipid hydroperoxides. Aldehydic phospholipids, as secondary products of peroxidation, were detected in oxidized membranes. EGP was the most prominent aldehydic phospholipid, while negligible amounts of aldehydic CGP were formed. This study indicates that the process of oxidation of individual phospholipids clearly differs among phospholipids and depends on the structure of each.     

New Geometric Isomers of Oxooctadecadienoate in Copper-catalyzed Decomposition Products of Linoleate Hydroperoxide

Methyl linoleate hydroperoxide produced by autoxidation was refluxed with 10^-4 M Cu-naphthenate in benzene. Two new geometrical isomers of oxooctadecadienoate (compounds I and II) were found in addition to the four known isomers. They were isolated by a Sephadex LH-20 column chromatography with chloroform-hexane (2:1) and purified by HPLC on Nucleosil ®100-5 and Zorbax ODS columns. UV, IR, MS, and ¹H-NMR spectra were measured. The geometry of conjugated dienes were assigned from the coupling constants of the olefinic protons. Compounds I and II were identified as 13-oxo-trans-9, cis-11- and 9-oxo-cis-10, trans-12-octadecadienoate, respectively. Each of them had a cis double bond adjacent to the oxo group. The hydroperoxides of the same geometry as compounds I and II were also detected in autoxidation products.     

Purification and characterization of tomato lipoxygenase

Lipoxygenase was partially purified (26-fold) from tomato (Lycopersicon esculentum) fruits by ammonium sulphate precipitation and hydrophobic chromatography, and further characterized by disc gel electrophoresis, chromatofocusing and M, determination. The enzyme had a pH optimum of 6.8, and K_m values for linoleic acid and linolenic acid of 1.42 and 2.60 mM, respectively. The pI was 6.3 and electrophoresis at pH 8.0 revealed a major lipoxygenase band at R_f 0.14. M, determination gave a value of 97 ± 2K. Incubation of linoleic acid with partially purified enzyme gave a mixture of linoleic hydroperoxides in which the ratio of the 9- to the 13-hydroperoxide isomer was 24:1. The major product was characterized as 9-hydroperoxyoctadeca-trans-10-cis-12-dienoic acid.