Isomerization of Oxidized Linoleate Metabolites with trans/cis Conjugated Diene Moiety to Those with trans/trans One in the Lipoxygenase/Linoleate/Hydrogen Polysulfide System

Abstract

Endogenous hydrogen polysulfides are radical scavengers, and the resulting thiyl radical may catalyze isomerization of the cis-double bond to a trans-double bond. This study examined whether oxidized linoleate species with trans/trans-conjugated diene moieties were generated in the 15-lipoxygenase/linoleate/hydrogen polysulfide system at a lower oxygen content. When 40 µL of 0.1 M phosphate buffer (pH 7.4) containing 1.0 mM linoleate, 1.0 µM soybean 15-lipoxygenase, and 100 µM sodium trisulfide was placed in a 0.6 mL polypropylene microtube for 1 h at 25 °C, the proportion of (E/E)-oxo-octadecadienoic acids (OxoODEs) content to the total OxoODEs content was estimated to be more than 80% (mol/mol). OxoODEs are generated through the pseudoperoxidase reaction of ferrous 15-lipoxygenase with hydroperoxy octadecadienoic acids (HpODEs), which are produced by the lipoxygenase reaction of ferric 15-lipoxygenase. The content of OxoODEs was positively correlated with the content of 9-HpODEs, indicating that 9-HpODEs production is involved in converting ferric 15-lipoxygenase to ferrous 15-lipoxygenase. Furthermore, when 40 µL of 0.1 M phosphate buffer (pH 7.4) containing 1.0 mM linoleate, 1.0 µM soybean 15-lipoxygenase, 100 µM sodium trisulfide, and nitroxyl radical (carbon-centered radical-trapping agent, 3-carbamoyl-2,2,5,5-tetramethyl-3-pyrrolin-N-oxyl (CmΔP)) was incubated in a 0.6 mL polypropylene microtube at room temperature, CmΔP-(E/Z)-ODEs were isomerized to CmΔP-(E/E)-ODEs in a time-dependent manner and this isomerization was inhibited by a radical scavenger, Trolox. The results indicate that thiyl radicals derived from hydrogen polysulfides isomerize trans/cis conjugated diene moiety to the trans/trans moiety.

INTRODUCTION

In organisms from plants to mammals, 12/15-lipoxygenases occur widely and constitutively. The enzymatic reaction of 12/15-lipoxygenases with polyunsaturated fatty acids as substrates produces several lipid mediators. Under aerobic conditions, ferric 12/15-lipoxygenases insert oxygen molecules into polyunsaturated fatty acids to produce hydroperoxy fatty acids with high stereospecificity and regiospecificity. In contrast, at a lower oxygen content, some of the resultant hydroperoxy fatty acids are converted to the corresponding oxo-fatty acids via ferrous 12/15-lipoxygenase through a pseudoperoxidase reaction.¹⁾ Oxo-fatty acids are peroxisome proliferator-activated receptor (PPAR) agonists, regulating lipid metabolism.²⁾ Generally, the conjugated diene moiety of oxo-fatty acids derived from hydroperoxy fatty acids with trans/cis (E/Z)-conjugated diene moiety is in the trans/cis form. Interestingly, oxo-octadecadienoic acids (OxoODEs) with trans/trans (E/E) conjugated diene moiety derived from linoleate possess strong PPAR agonist activity,^3,4) and especially (E/E)-OxoODEs appear to be present in tomato juice.⁵⁾ However, there are no reports on reaction systems suggesting the production of (E/E)-OxoODEs in vivo.

Hydrogen sulfide (pK_a1 7.0, pK_a2 17.2; [HS⁻]/[H₂S] = 2.5 at pH 7.4)⁶⁾ is a gaseous substance produced constitutively in organisms such as bacteria, plants, and mammals. Hydrogen sulfide is a gaseous mediator, especially in mammals (see review⁷⁾), and chemically acts as a radical scavenger; therefore, hydrogen sulfide is converted to hydrogen polysulfides, including hydrogen disulfide (pK_a1 5.0, pK_a2 9.7; [S₂²⁻]/[HS₂⁻] = 0.0050 at pH 7.4)⁶⁾ and hydrogen trisulfide (pK_a1 4.2, pK_a2 7.5; [S₃²⁻]/[HS₃⁻] = 0.79 at pH 7.4)⁶⁾ in cells expressing physiological radical-producing systems.^8–10) Koike et al.¹¹⁾ reported that both hydrogen disulfide and hydrogen trisulfide exist in brain neurons expressing neuronal nitric oxide synthetase. Thus, hydrogen polysulfides are endogenous bioactive substances that maintain homeostasis in vivo. Hydrogen polysulfides have a stronger radical scavenging activity than hydrogen sulfide¹²⁾; thus, they are easily converted to the corresponding thiyl radicals by one-electron oxidation or one-electron reduction in radical-generating systems.

  

(1)

  

(2)

Thiyl radicals catalyze the cis–trans isomerization of double bonds in unsaturated fatty acids.^13–17) Double bond consists of both σ- and π-bonds. Thiyl radicals attack the cis-type double bond and reversibly cleave the π-bond, resulting in conversion to a thermodynamically stable trans-type double bond. In our previous study,¹⁾ we revealed that the pseudoperoxidase reaction of ferrous 15-lipoxygenase with hydroperoxy fatty acids as a substrate at a lower oxygen content produces various lipid radical species. If hydrogen polysulfides coexist in this pseudoperoxidase reaction system, it is likely that the hydrogen polysulfide-derived thiyl radicals convert the trans/cis conjugated diene moiety of the resultant oxidized fatty acids to trans/trans one. However, it is unclear whether hydrogen polysulfide-derived thiyl radicals are generated via the reaction of hydrogen polysulfides with lipid radical species including oxygen-centered radical and carbon-centered radical. In this study, we examined the isomerization of trans/cis conjugated diene moiety into trans/trans conjugated diene moiety of the reaction products in the 15-lipoxygenase/linoleate system at a lower oxygen content in the presence of hydrogen polysulfides.

MATERIALS AND METHODS

Materials

Soybean 15-lipoxygenase (Type I-b; activity, 70800 units/mg; molecular size, 108 kDa), linoleic acid, and 3-carbamoyl-2,2,5,5-tetramethyl-3-pyrrolin-N-oxyl (CmΔP) were purchased from Sigma-Aldrich Co. (St. Louis, MO, U.S.A.). 13-Hydroperoxy-(9Z,11E)-octadecadienoic acid (13-(E/Z)-HpODE), 13-hydroperoxy-(9E,11E)-octadecadienoic acid (13-(E/E)-HpODE), 9-hydroperoxy-(10E,12Z)-octadecadienoic acid (9-(E/Z)-HpODE), 9-hydroperoxy-(10E,12E)-octadecadienoic acid (9-(E/E)-HpODE), 13-oxo-(9Z,11E)-octadecadienoic acid (13-(E/Z)-OxoODE), 9-oxo-(10E,12Z)-octadecadienoic acid (9-(E/Z)-OxoODE), 13-hydroxy-(9Z,11E)-octadecadienoic acid (13-(E/Z)-HODE), 13-hydroxy-(9E,11E)-octadecadienoic acid (13-(E/E)-HODE), 9-hydroxy-(10E,12Z)-octadecadienoic acid (9-(E/Z)-HODE), and 9-hydroxy-(10E,12E)-octadecadienoic acid (9-(E/E)-HODE) were purchased from Larodan (Stockholm, Sweden). Sodium disulfide and sodium trisulfide were purchased from Dojindo Laboratories (Kumamoto, Japan). TSKgel ODS-80Ts QA (2.0 mm I.D. × 150 mm; particle size, 5 µm) and TSKguardgel ODS-80Ts were purchased from Tosoh Corp. (Tokyo, Japan). All other chemicals used were of reagent grade.

HPLC Analyses of HpODEs, OxoODEs, and HODEs

13-(E/Z)-HpODE, 9-(E/Z)-HpODE, 13-(E/E)-HpODE, 9-(E/E)-HpODE, 13-(E/Z)-HODE, 9-(E/Z)-HODE, 13-(E/E)-HODE, 9-(E/E)-HODE,13-(E/Z)-OxoODE, 9-(E/Z)-OxoODE, 13-(E/E)-OxoODE, and 9-(E/E)-OxoODE were analyzed via reverse-phase HPLC with dual-wavelength UV detection and photodiode array detection. The HPLC assembly consisted of an HPLC pump (515; Waters, MA, U.S.A.), Rheodyne sample injector (7725; Idex Health & Science, CA, U.S.A.), temperature control module (Waters), column heater module (Waters), dual-λ absorbance detector (2487; Waters), data station (µ7; SIC, Tokyo, Japan), and a photodiode array detector (Hewlett Packard, CA, U.S.A.). The chromatographic conditions for the analyses of these substances were as follows: column, two TSKgel ODS-80Ts QA (2.0 mm I.D. × 150 mm) connected in series; eluent, 0.05% formic acid containing 50% acetonitrile; flow rate, 0.15 mL/min; column temperature, 35 °C; detection wavelength, 234 nm (for 13-(E/Z)-HpODE, 9-(E/Z)-HpODE, 13-(E/E)-HpODE, 9-(E/E)-HpODE, 13-(E/Z)-HODE, 9-(E/Z)-HODE, 13-(E/E)-HODE, and 9-(E/E)-HODE) and 280 nm (for 13-(E/Z)-OxoODE, 9-(E/Z)-OxoODE, 13-(E/E)-OxoODE, and 9-(E/E)-OxoODE). In this study, the relative abundances of these analytes in the reaction solutions are presented in the peak area because commercially available standard substances were of qualification grade and not quantification grade.

Effects of Sodium Polysulfides on the Isomerization of the Conjugated Diene Moiety in HpODEs, OxoODEs, and HODEs Produced in the Lipoxygenase/Linoleate System at a Lower Oxygen Content

Twenty microliters of 2 mM linoleic acid emulsion in 0.1 M phosphate buffer (pH 7.4) containing 2% ethanol were mixed with 10 µL of 4.0 μM soybean lipoxygenase-1 in 0.1 M phosphate buffer (pH 7.4) and 10 µL of Na₂S₂ or Na₂S₃ in 0.1 M phosphate buffer (pH 7.4) in a polypropylene microtube (inner volume, 0.6 mL). Subsequently, the solution was kept at room temperature (25–28 °C) for the durations indicated in the figure legends. A portion of the reaction solution (5 µL) was subjected to HPLC analysis.

Direct Reaction of Isolated 13-(E/Z)-HpODE with Na₂S₂

Thirty microliters of 67 μM 13-(E/Z)-HpODE in 0.1 M phosphate buffer (pH 7.4) containing 2% ethanol was mixed with 10 µL of 200 μM Na₂S₂ in 0.1 M phosphate buffer (pH 7.4) in a polypropylene microtube (inner volume, 0.6 mL). Subsequently, the solution was incubated at room temperature (25–28 °C) for the durations indicated in the figure legends. A portion of the reaction solution (5 µL) was subjected to HPLC analysis.

HPLC Analyses of Fatty Acid Allyl Radical−CmΔP Adducts

In our previous study, we established an HPLC method to quantify fatty acid allyl radical CmΔP adducts simultaneously.^18–20) The chromatographic conditions for the analysis of these substances were as follows: column, TSKgel ODS-80Ts QA (2.0 mm I.D. × 150 mm); eluent, 0.05% formic acid containing 50% acetonitrile; flow rate, 0.3 mL/min; column temperature, 35 °C; detection at 234 nm for fatty acid allyl radical−CmΔP adducts.

Effects of Sodium Polysulfides on the Isomerization of the Conjugated Diene Moiety in Fatty Acid Allyl Radical−CmΔP Adducts

Ten microliters of 4 mM linoleic acid emulsion in 0.1 M phosphate buffer (pH 7.4) containing 4% ethanol were mixed with 10 µL of 4.0 mM CmΔP in 0.1 M phosphate buffer (pH 7.4), 10 µL of sodium polysulfide in 0.1 M phosphate buffer (pH 7.4), and 10 µL of 4.0 μM soybean 15-lipoxygenase in 0.1 M phosphate buffer (pH 7.4) in series in a polypropylene microtube (inner volume, 0.6 mL), and the solution was left at room temperature (25–28 °C) for the durations indicated in the figure legends. The reaction mixture was then subjected to HPLC analysis.

Statistical Analysis

Data are expressed as mean ± standard deviation (S.D.). Statistical analyses were performed using Dunnett’s test for multiple comparisons. The analysis was carried out using IBM SPSS statistics 25 (IBM Japan, Ltd., Tokyo, Japan).

RESULTS

Chromatographic Quantification of Linoleate Oxidized Species Produced in the Lipoxygenase/Linoleate System Containing Sodium Polysulfides at a Lower Oxygen Content

In our previous study,¹⁾ we elucidated that not only linoleate hydroperoxides (13-HpODE and 9-HpODE) but also OxoODEs (13-OxoODE and 9-OxoODE) were produced in the soybean 15-lipoxygenase (1.0 μM)/linoleate (1.0 mM) system through lipoxygenase and pseudoperoxidase reactions, respectively. In this reaction system (40 µL of reaction solution in a polypropylene microtube (inner volume, 0.6 mL)), the dissolved oxygen content in the reaction solution is lowered within a few minutes via the dioxygenation of the linoleate; subsequently, oxygen molecules in the air phase diffuse into the reaction solution.^14,16) In the present study, this state was defined as “the lower oxygen content.” The possible reaction pathways in the lipoxygenase/linoleate system at a lower oxygen content are shown in Fig. 1. Both 13-OxoODE and 13-HODE are produced from alkoxyl radicals as intermediates generated through the pseudoperoxidation of 13-HpODE by ferrous 15-lipoxygenase as follows:

  

(3)

  

(4)

Fig. 1. Possible Reaction Paths in the Lipoxygenase/Linoleate System at a Lower Oxygen Content

Linoleate is converted to linoleate allyl radicals via the withdrawal of allylic hydrogen by ferric lipoxygenase. At a lower oxygen contents, linoleate allyl radical-ferrous lipoxygenase complex or linoleate peroxyl radical-ferrous lipoxygenase must be dissociated into ferrous lipoxygenase and linoleate-derived radicals. Concomitantly, ferrous lipoxygenase converts (E/Z)-HpODEs into (E/Z)-linoleate alkoxyl radicals via a pseudoperoxidase reaction. A portion of the liberated linoleate alkoxyl radicals is converted to OxoODEs through one-electron oxidation, whereas the other is converted to HODEs and linoleate epoxy-hydroperoxide through one-electron reduction.

The reaction products formed by the reaction of soybean 15-lipoxygenase with linoleate in the presence of sodium polysulfide were quantified by HPLC using a dual-wavelength UV-Vis detector. Soybean 15-lipoxygenase (1.0 μM) and linoleate (1.0 mM) were incubated in 100 mM phosphate buffer solution (pH 7.4) in the presence or absence of 100 μM sodium disulfide for 60 min at room temperature, and the reaction solutions were subjected to HPLC analysis. The chromatograms are shown in Fig. 2. In the absence of sodium disulfide, the stereospecificity of the conjugated diene moieties of the products was almost the E/Z-configuration (data not shown), whereas products with the E/E-configuration were formed in the presence of sodium disulfide.

Fig. 2. Chromatographic Analyses of HpODEs, HODEs, and OxoODEs

The mixture solution of 1.0 mM linoleic acid, 1.0 μM soybean lipoxygenase-1, and 100 μM sodium disulfide in 0.1 M phosphate buffer (pH 7.4) in a polypropylene microtube (inner volume, 0.6 mL) was kept at room temperature (25–28 °C) for 60 min. The reaction mixture was then subjected to HPLC analysis. HpODEs and HODEs were detected using a dual-λ absorbance detector at 234 nm (A) and OxoODEs were detected at 280 nm (B). In Inset B, two overlapping peaks were resolved into individual peaks using data station μ7.

Effects of Sodium Polysulfides on the Production of the Linoleate Oxidized Species Produced in the Lipoxygenase/Linoleate System at a Lower Oxygen Content

It is generally known that the oxygen content of water is approximately 200–250 μM. Therefore, when linoleate at a higher content than that of oxygen is incubated with 15-lipoxygenase, the oxygen molecules in the reaction solution are nearly consumed. Figure 3 shows the time course of the linoleate-oxidized species produced in the lipoxygenase (1.0 μM)/linoleate (1.0 mM) system containing 20 μM sodium disulfide or 20 μM sodium trisulfide. As shown in Fig. 3A, both 13-(E/Z)-HpODE and 13-(E/E)-HpODE reached plateaus within 5 min. In contrast, the OxoODE content increased up to 60–120 min, suggesting that the production of OxoODEs proceeds at a lower oxygen content.

Fig. 3. Time Course of the Contents of Linoleate-Oxidized Species in the Lipoxygenase/Linoleate System

The mixture solution of 1.0 mM linoleic acid, 1.0 μM soybean lipoxygenase-1, and 20 μM sodium polysulfide in 0.1 M phosphate buffer (pH 7.4) was kept in a polypropylene microtube (inner volume, 0.6 mL) at room temperature (25–28 °C) for the time intervals indicated in the figure. The reaction mixture was then subjected to HPLC analysis. The contents of 13-(E/Z)-HpODE and 13-(E/E)-HpODE reached a plateau within 5 min. In contrast, (E/Z)-OxoODEs, (E/Z)-HODEs, (E/E)-OxoODEs, and (E/E)-HODEs increased remarkably from 30 to 120 min. The results are presented as the mean ± S.D. of triplicate experiments.

The production of oxidized linoleate species in the lipoxygenase (1.0 μM)/linoleate (1.0 mM) system containing sodium disulfide or sodium trisulfide at concentrations ranging from 10 to 100 μM is shown in Fig. 4. The production of almost all oxidized linoleate species with trans/trans conjugated diene moieties was dependent on the sodium polysulfide content, as the reaction was accelerated in the presence of sodium polysulfides. Interestingly, the increase in (E/E)-OxoODEs with increasing sodium polysulfide content was accompanied by a decrease in (E/Z)-OxoODEs (Figs. 4B, E). It is noted that hydrogen polysulfides can slightly reduce HpODEs to HODEs in the polysulfide content dependent manner.²¹⁾

Fig. 4. Effects of Sodium Polysulfides on the Production of Linoleate-Oxidized Species in the Lipoxygenase/Linoleate System

The mixture solution of 1.0 mM linoleic acid, 1.0 μM soybean lipoxygenase-1, and sodium polysulfide (10, 20, 40, 70, or 100 μM) in 0.1 M phosphate buffer (pH 7.4) was kept in a polypropylene microtube (inner volume, 0.6 mL) at room temperature (25–28 °C) for 60 min. The reaction mixture was then subjected to HPLC analysis. Almost all the linoleate-oxidized species with trans/trans-conjugated diene moieties increased in a sodium polysulfide content-dependent manner. The results are presented as the mean ± S.D. of triplicate experiments.

Possibility of Isomerization of the Isolated 13-(E/Z)-HpODE to 13-(E/E)-HpODE through the Direct Reaction with Sodium Disulfide

When 50 μM 13-(E/Z)-HpODE was incubated with 50 μM sodium disulfide in 100 mM phosphate buffer solution (pH 7.4) at room temperature, a slight decrease was observed in the 13-(E/Z)-HpODE content at the early stage of the reaction, but no significant increase was observed in the 13-(E/E)-HpODE content (Fig. 5). Therefore, sodium disulfide could not isomerize 13-(E/Z)-HpODE to 13-(E/E)-HpODE.

Fig. 5. Reaction of Sodium Disulfide with Isolated 13-(E/Z)-HpODE

Phosphate buffer solution (100 mM, pH 7.4) containing 50 μM 13-(E/Z)-HpODE and 50 μM sodium disulfide was incubated at room temperature. The solution was then subjected to HPLC analysis. Each product was quantified using HPLC. Isomerization of 13-(E/Z)-HpODE to 13-(E/E)-HpODE via a direct reaction with sodium disulfide was not observed. The results are presented as the mean ± S.D. of triplicate experiments.

Trapping Analyses of Carbon-Centered Radicals Produced in the Lipoxygenase/Linoleate/Sodium Polysulfide System at a Lower Oxygen Content via Nitroxyl Radical, CmΔP

In our previous study,^13–15) we elucidated that at a lower oxygen content, nitroxyl radicals can trap the carbon-center radical species complexed with 15-lipoxygenase, including the linoleate allyl radical with ferrous 15-lipoxygenase and linoleate epoxyallyl radical with ferric 15-lipoxygenase. The possible reaction pathways in the lipoxygenase/linoleate/CmΔP system at a lower oxygen content are shown in Fig. 6. It should be noted that the linoleate allyl radical-CmΔP adducts in the lipoxygenase/linoleate/CmΔP system were 13-CmΔP-(E/Z)-ODE and 9-CmΔP-(E/Z)-ODE. The linoleate allyl radical-CmΔP adducts were detected by HPLC with UV detection at 234 nm (Fig. 7A). In contrast, in the lipoxygenase/linoleate/CmΔP system in the presence of 100 μM sodium disulfide, not only CmΔP-(E/Z)-ODEs but also CmΔP-(E/E)-ODEs were detected by HPLC (Fig. 7B). 9-CmΔP-(E/Z)-ODE was completely separated from 9-CmΔP-(E/E)-ODE, whereas 13-CmΔP-(E/Z)-ODE and 13-CmΔP-(E/E)-ODE eluted at the same elution position.¹⁹⁾ Time–courses of the content of 9-CmΔP-(E/Z)-ODE and 9-CmΔP-(E/E)-ODE are shown in Fig. 8A. The correlation between the 9-CmΔP-(E/E)-ODE content and 9-CmΔP-(E/Z)-ODE content after the enzyme reaction reached a plateau is shown in Fig. 8B. This finding indicated that 9-CmΔP-(E/Z)-ODE should be converted to 9-CmΔP-(E/E)-ODE in a time-dependent manner. The effects of the sodium polysulfide content on the proportion of 9-CmΔP-(E/E)-ODE to 9-CmΔP-ODEs are shown in Fig. 8C. Furthermore, the presence of Trolox did not influence both the lipoxygenase and pseudoperoxidase reactions at all (Fig. 9A), whereas this conversion was significantly inhibited in the presence of the representative radical scavenger Trolox (Fig. 9B).

Fig. 6. Scheme of One Electron Redox Cycle in the Fatty Acid/Fatty Acid Hydroperoxide/Lipoxygenase System Containing CmΔP

CmΔP directly attacks the fatty acid-derived allyl radical and fatty acid hydroperoxide-derived epoxyallyl radicals at the reaction site of the enzyme.

Fig. 7. Typical Chromatograms of the Products Generated in the Lipoxygenase/Linoleate/CmΔP System in the Presence or Absence of Sodium Disulfide

In a 0.6 mL polypropylene microtube, 0.1 M phosphate buffer solution (pH 7.4) containing 1.0 mM CmΔP, 1.0 mM linoleic acid, and 1.0 μM soybean 15-lipoxygenase was kept for 60 min at room temperature in the presence (B) or absence (A) of 100 μM sodium disulfide, and the reaction solution was subjected to HPLC. The amounts of 13-CmΔP-ODEs and 9-CmΔP-ODEs were almost equal.

Fig. 8. Effects of Sodium Polysulfides on the Isomerization of Conjugated Diene Moiety of Linoleate Allyl Radical-CmΔP Adducts

A, The mixture solution of 1.0 mM linoleic acid, 1.0 μM soybean lipoxygenase-1, 1.0 mM CmΔP, and 100 μM sodium trisulfide in 0.1 M phosphate buffer (pH 7.4) was kept in a polypropylene microtube (inner volume, 0.6 mL) at room temperature (25–28 °C) for the time intervals indicated in the figure. The correlation between the content of 9-CmΔP-(E/E)-ODE and that of 9-CmΔP-(E/Z)-ODE is shown in panel B. Each number in the panels indicates the reaction time. C, The mixture solution of 1.0 mM CmΔP, 1.0 mM linoleic acid, 1.0 μM soybean lipoxygenase-1, and sodium polysulfide (10, 20, 40, 70, or 100 μM) in 0.1 M phosphate buffer (pH 7.4) was kept in a polypropylene microtube (inner volume, 0.6 mL) at room temperature (25–28 °C) for 60 min. The proportion of the (E/E) form was calculated from the contents of 9-CmΔP-(E/E)-ODE and 9-CmΔP-(E/Z)-ODE. Each product was quantified using HPLC. The results are presented as mean ± S.D. obtained from triplicate experiments.

Fig. 9. Effects of Trolox on the Production of 13-CmΔP-ODEs (A) and the Isomerization of 9-CmΔP-(E/Z)-ODE to 9-CmΔP-(E/E)-ODE (B) in the 15-Lipoxygenase/Linoleate/CmΔP/Sodium Disulfide System

The mixture solution of 1.0 mM linoleic acid, 1.0 μM soybean lipoxygenase-1, 1.0 mM CmΔP, and 100 μM sodium disulfide in 0.1 M phosphate buffer (pH 7.4) was kept in a polypropylene microtube (inner volume, 0.6 mL) at room temperature (25–28 °C) for 60 min in the presence or absence of 500 μM Trolox. The proportion of the (E/E) form was calculated from the contents of 9-CmΔP-(E/E)-ODE and 9-CmΔP-(E/Z)-ODE. Each product was quantified using HPLC. The results are presented as mean ± S.D. obtained from triplicate experiments. Asterisks indicate significant differences compared with the control experiment (* p < 0.01).

DISCUSSION

The first question is how oxidized linoleate derivatives with E/E-conjugated diene moieties are produced in the lipoxygenase/linoleate/sodium polysulfide system. The possible mechanisms are as follows: (1) the stereospecificity of the lipoxygenase reaction is lowered, producing (E/E)-HpODEs or (2) isomerization of the resultant oxidized linoleate derivatives with E/Z-conjugated diene moieties to those with E/E-conjugated diene moieties. The rationale for pathway (1) is as follows: soybean 15-lipoxygenase has four free sulfhydryl groups, and chemical modification of these groups has been reported to result in the loss of stereospecificity in the lipoxygenase reaction.²²⁾ It should be noted that hydrogen polysulfides, which are sulfane sulfur donors, rapidly donate sulfane sulfur to the sulfhydryl groups in proteins.²³⁾

In general, (E/E)-HpODEs are produced by the radical chain reaction of linoleate. During this process, free linoleate allyl radicals are produced as intermediates, resulting in a thermodynamically stable E/E-conjugated diene structure. In contrast, ferric 15-lipoxygenase reacts with linoleate to form a linoleate allyl radical-ferrous lipoxygenase complex as an intermediate. Stereospecificity is maintained by strictly restricting the movement of the carbon skeleton of the linoleate radical at the binding site of ferrous lipoxygenase. If the binding of the linoleate ally radical to ferrous lipoxygenase is loosened, the trans/cis-conjugated diene moiety should change to a thermodynamically stable trans/trans conjugated diene moiety, resulting in the (E/E)-HpODEs production. According to this hypothesis, the stereospecificity of the conjugated diene moieties of HpODEs may reflect that of OxoODEs. Interestingly, as shown in Fig. 3, despite the higher content of 13-(E/Z)-HpODE than 13-(E/E)-HpODE, the 13-(E/E)-OxoODE content was greater than the 13-(E/Z)-OxoODE content. However, it is unlikely that the substrate specificity of ferrous lipoxygenase for 13-(E/E)-HpODEs is superior to that for 13-(E/Z)-HpODEs.²⁴⁾

Furthermore, in order to clarify the conjugated diene structure of the linoleate allyl radical complexed with ferrous lipoxygenase, we performed trapping experiments using the nitroxyl radical (CmΔP), which can trap carbon-centered radicals. On the assumption that the linoleate allyl radical with trans/trans-conjugated diene loosely binds to ferrous lipoxygenase, the trapping adducts must be chemically stable CmΔP-(E/E)-ODEs. However, as shown in Fig. 8, the formation of CmΔP-(E/Z)-ODEs preceded the formation of CmΔP-(E/E)-ODEs in the initial stage of the reaction, followed by the isomerization of CmΔP-(E/Z)-ODEs to CmΔP-(E/E)-ODEs. This isomerization is significantly inhibited by Trolox, indicating the involvement of a radical reaction. Sulfur-centered radicals (thiyl radicals) have long been known to catalyze isomerization of the cis double bond to the trans form.^13–17) Taken together, we propose that oxidized (E/Z)-linoleate derivatives produced in the lipoxygenase/linoleate system may be converted to (E/E)-isomers by polysulfide-derived thiyl radicals, which are produced by the reaction of hydrogen polysulfides with fatty acid-derived radicals.

The second question is how ferric lipoxygenase is converted to ferrous lipoxygenase at a lower oxygen content (Fig. 1). OxoODEs are produced through the pseudoperoxidase reaction by ferrous lipoxygenase using HpODEs as a substrate. Therefore, we evaluated the correlations between the OxoODE content and HpODE content in the lipoxygenase/linoleate/sodium polysulfide system using the analytical data in Fig. 3. As shown in Fig. 10, the OxoODE content (both 13-OxoODEs and 9-OxoODEs) correlated with 9-HpODEs content but not with 13-HpODEs content. Based on this, we propose the following hypotheses: (1) At a lower oxygen content, the regiospecificity of the insertion of oxygen molecules may be lowered, so that not only linoleate 13-peroxyl radicals but also linoleate 9-peroxyl radicals are produced at the reaction site of ferrous-lipoxygenase. (2) An electron seems to be transferred from ferrous iron to the peroxyl radical at the 13-position of linoleate but not to that at the 9-position. (3) The linoleate 9-peroxyl radical dissociated from ferrous lipoxygenase is reduced to 9-HpODE through one-electron reduction by hydrogen polysulfides, resulting in the generation of polysulfide-derived thiyl radicals (Fig. 11).

Fig. 10. Correlations between the Contents of Regioisomers of OxoODEs and Those of Regioisomers of HpODEs

Each panel was made by using the quantification data of HpODEs and OxoODEs in the lipoxygenase (1.0 μM)/linoleate (1.0 mM)/Na₂S₂ (20 μM) system shown in Fig. 3 (A, B, D, E).

Fig. 11. Possible Reaction Path for Conversion of Ferric 15-Lipoxygenase to Ferrous One through the Lipoxygenase Reaction with Linoleate at a Lower Oxygen Content

It remains unclear whether the hydrogen polysulfide scavenges the linoleate 9-peroxyl radical on the enzyme or liberated linoleate 9-peroxyl radical from the enzyme.

CONCLUSION

The following mechanism can be considered for (E/E)-OxoODEs production in the 15-lipoxygenase reaction, using linoleate as a substrate in the presence of hydrogen polysulfides. (1) At a lower oxygen contents, linoleate 13-peroxyl radicals and linoleate 9-peroxyl radicals complexed with ferrous lipoxygenase are produced. Subsequently, the linoleate 9-peroxyl radical-ferrous lipoxygenase complex dissolves, and the resultant linoleate 9-peroxyl radical is scavenged by the hydrogen polysulfide, resulting in the generation of a thiyl radical. (2) A portion of (E/Z)-HpODEs is converted to (E/Z)-OxoODEs through a pseudoperoxidase reaction via the resultant ferrous lipoxygenase. (3) (E/Z)-OxoODEs are isomerized to (E/E)-OxoODEs through a catalytic reaction via the polysulfide-derived thiyl radical. In conclusion, it is likely that the conjugated polyene moiety in biological substances also isomerizes to a thermodynamically stable conjugated polyene structure in the presence of the polysulfide-derived thiyl radical.

Acknowledgments

This work was financially supported by JSPS KAKENHI (Grant Nos. JP18K11015 and JP21K11719).

Conflict of Interest

The authors declare no conflict of interest.

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