Glutathionylated 4-hydroxy-2-(E)-alkenal enantiomers in rat organs and their contributions toward the disposal of 4-hydroxy-2-(E)-nonenal in rat liver

The major route for elimination of 4-hydroxy-2-(E)-nonenal (4-HNE) has long been considered to be through glutathionylation and eventual excretion as a mercapturic acid conjugate. To better quantitate the glutathionylation process, we developed a sensitive LC–MS/MS method for the detection of glutathione (GSH) conjugates of 4-hydroxy-2-(E)-alkenal enantiomers having a carbon skeleton of C5 to C12. The newly developed method enabled us to quantify 4-hydroxy-2-(E)-alkenal–glutathione diastereomers in various organs, i.e., liver, heart, and brain. We identified the addition of iodoacetic acid as a critical step during sample preparation to avoid an overestimation of glutathione–alkenal conjugation. Specifically, we found that in the absence of a quenching step reduced GSH and 4-hydroxy-2-(E)-alkenals react very rapidly during the extraction and concentration steps of sample preparation. Rat liver perfused with d₁₁-4-hydroxy-2-(E)-nonenal (d₁₁-4-HNE) revealed enantioselective conjugation with GSH and transportation out of the liver. In the d₁₁-4-HNE-perfused rat livers, the amount of d₁₁-(S)-4-HNE–GSH released from the rat liver was higher than that of d₁₁-(R)-4-HNE–GSH, and more d₁₁-(R)-4-HNE–GSH than d₁₁-(S)-4-HNE–GSH remained in the perfused liver tissues. Overall, the glutathionylation pathway was found to account for only 8.7% of the disposition of 4-HNE, whereas catabolism to acetyl-CoA, propionyl-CoA, and formate represented the major detoxification pathway.     

Identification and absolute configuration of dihydroxy-arachidonic acids formed by oxygenation of 5S-HETE by native and aspirin-acetylated COX-2

Biosynthesis of the prostaglandin endoperoxide by the cyclooxygenase (COX) enzymes is accompanied by formation of a small amount of 11R-hydroxyeicosatetraenoic acid (HETE), 15R-HETE, and 15S-HETE as by-products. Acetylation of COX-2 by aspirin abrogates prostaglandin synthesis and triggers formation of 15R-HETE as the sole product of oxygenation of arachidonic acid. Here, we investigated the formation of by-products of the transformation of 5S-HETE by native COX-2 and by aspirin-acetylated COX-2 using HPLC-ultraviolet, GC-MS, and LC-MS analysis. 5S,15S- dihydroxy (di)HETE, 5S,15R-diHETE, and 5S,11R-diHETE were identified as by-products of native COX-2, in addition to the previously described di-endoperoxide (5S,15S-dihydroxy-9S,11R,8S,12S-diperoxy-6E,13E-eicosadienoic acid) as the major oxygenation product. 5S,15R-diHETE was the only product formed by aspirin-acetylated COX-2. Both 5,15-diHETE and 5,11-diHETE were detected in CT26 mouse colon carcinoma cells as well as in lipopolysaccharide-activated RAW264.7 cells incubated with 5S-HETE, and their formation was attenuated in the presence of the COX-2 specific inhibitor, NS-398. Aspirin-treated CT26 cells gave 5,15-diHETE as the most prominent product formed from 5S-HETE. 5S,15S-diHETE has been described as a product of the cross-over of 5-lipoxygenase (5-LOX) and 15-LOX activities in elicited rat mononuclear cells and human leukocytes, and our studies implicate cross-over of the 5-LOX and COX-2 pathways as an additional biosynthetic route.     

Effects of epoxymethano analogues of prostaglandin endoperoxides on aggregation,on release of 5-hydroxytryptamine and on the metabolism of 3′,5′-cyclic AMP and cyclic GMP in human platelets

The effects on human platelets of two synthetic analogues of prostaglandin endoperoxides were examined in order to explore the relationship between aggregation and prostaglandin and cyclic nucleotide metabolism, and to help elucidate the role of the natural endoperoxide intermediates in regulating platelet function.Both analogues (Compound I, (15S)-hydroxy-9α,11α-(epoxymethano)-prosta-(5Z,13E)-dienoic acid, and Compound II, (15S)-hydroxy-11α,9α-(epoxymethano)-prosta-(5Z,13E)-dienoic acid) caused platelets to aggregate, an effect which could be inhibited by prostaglandin E₁ but not by indomethacin. Compound II produced primary, reversible aggregation at concentrations which did not induce release of 5-hydroxytryptamine. Production of thromboxane B₂ and malonyldialdehyde was monitored as an index of endogenous production of prostaglandin endoperoxides and thromboxane A₂ and were increased after incubation of human platelets with thrombin, collagen or arachidonic acid. However, neither malonydialdehyde nor thromboxane B₂ levels were significantly influenced by the endoperoxide analogues. Both analogues produced a small elevation of adenylate cyclase activity in platelet membranes and of cyclic AMP content in intact platelets, but neither had any modifying effect on the much greater stimulation of adenylate cyclase and cyclic AMP levels by prostaglandin E₁. Of all the aggregating agents tested, only arachidonic acid produced any significant increase in platelet cyclic GMP levels.These results suggest that the epoxymethano analogues of prostaglandin endoperoxides induce platelet aggregation independently of thromboxane biosynthesis and without inhibiting adenylate cyclase or lowerin platelet cyclic AMP levels. They therefore differ from better known aggregating agents such as ADP, epinephrine and collagen, which increase thromboxane A₂ production and reduce cyclic AMP levels, at least in platelets previously exposed to prostaglandin E₁.     

Arachidonate 15-lipoxygenase in human corneal epithelium and 12- and 15-lipoxygenases in bovine corneal epithelium: Comparison with other bovine 12-lipoxygenase

Lipoxygenases of bovine and human corneal epithelia were investigated. The bovine epithelium contained an arachidonate 12-lipoxygenase and a 15-lipoxygenase. The 12-lipoxygenase was found in the microsomal fraction, while the 15-lipoxygenase was mainly present in the cytosol (100 000 × g supernatant). 12S-Hydroxyeicosatetraenoic acid (12S-HETE) and 15S-hydroxyeicosa-tetraenoic acid (15S-HETE) were identified by GC-MS and chiral HPLC. BW A4C, an acetohydroxamic acid lipoxygenase inhibitor, reduced the biosynthesis of 12S-HETE and 15S-HETE by over 90% at 10 μ M. IC₅₀ for the 12-lipoxygenase was 0.3 μM. The bovine corneal 12-lipoxygenase was compared with the 12-lipoxygenases of bovine platelets and leukocytes. All three enzymes metabolized ¹⁴C-labelled linoleic acid and α-linolenic acid poorly (5–16%) in comparison with [^l4C]arachidonic acid. [¹⁴C]Docosahexaenoic acid and [¹⁴C]4,7,10,13,16-docosapentaenoic acid appeared to be less efficiently converted by the corneal enzyme than by the platelet and leukocyte enzymes. Immunohistochemical analysis of the bovine corneal epithelium using a polyconal antibody against porcine leukocyte 12-lipoxygenase gave positive staining. The cytosol of human corneal epithelium converted [¹⁴C]arachidonic acid to one prominent metabolite. The product co-chromatographed with 15S-HETE on reverse phase HPLC, straight phase HPLC and chiral HPLC. Our results suggest that human corneal epithelium contains a 15-lipoxygenase and that bovine corneal epithelium contains both a 15-lipoxygenase and a 12-lipoxygenase. The corneal 12-lipoxygenase appears to differ catalytically from earlier described bovine 12-lipoxygenases.     

COX-2-dependent and -independent biosynthesis of dihydroxy-arachidonic acids in activated human leukocytes

Biosynthesis of 5,15-dihydroxyeicosatetraenoic acid (5,15-diHETE) in leukocytes involves consecutive oxygenation of arachidonic acid by 5-lipoxygenase (LOX) and 15-LOX in either order. Here, we analyzed the contribution of cyclooxygenase (COX)-2 to the biosynthesis of 5,15-diHETE and 5,11-diHETE in isolated human leukocytes activated with lipopolysaccharide and calcium ionophore A23187. Transformation of arachidonic acid was initiated by 5-LOX providing 5S-HETE as a substrate for COX-2 forming 5S,15S-diHETE, 5S,15R-diHETE, and 5S,11R-diHETE as shown by LC/MS and chiral phase HPLC analyses. The levels of 5,15-diHETE were 0.45 ± 0.2 ng/10⁶ cells (mean ± SEM, n = 6), reaching about half the level of LTB₄ (1.3 ± 0.5 ng/10⁶ cells, n = 6). The COX-2 specific inhibitor NS-398 reduced the levels of 5,15-diHETE to below 0.02 ng/10⁶ cells in four of six samples. Similar reduction was achieved by MK-886, an inhibitor of 5-LOX activating protein but the above differences were not statistically significant. Aspirin treatment of the activated cells allowed formation of 5,15-diHETE (0.1 ± 0.05 ng/10⁶ cells, n = 6) but, as expected, abolished formation of 5,11-diHETE. The mixture of activated cells also produced 5S,12S-diHETE with the unusual 6E,8Z,10E double bond configuration, implicating biosynthesis by 5-LOX and 12-LOX activity rather than by hydrolysis of the leukotriene A₄-epoxide. Exogenous octadeuterated 5S-HETE and 15S-HETE were converted to 5,15-diHETE, implicating that multiple oxygenation pathways of arachidonic acid occur in activated leukocytes. The contribution of COX-2 to the biosynthesis of dihydroxylated derivatives of arachidonic acid provides evidence for functional coupling with 5-LOX in activated human leukocytes.     

Cloning and expression of three lipoxygenase genes from liverwort,Marchantia polymorpha L., in Escherichia coli

Three genes homologous to plant lipoxygenase genes were identified from the EST libraries of Marchantia polymorpha, in order to clarify the function of LOXs in bryophytes. Full-length genes were isolated using 5′- and 3′-RACE methods and named MpLOX1, MpLOX2, and MpLOX3, respectively. To investigate the enzymatic activities of liverwort LOXs, recombinant MpLOX1, MpLOX2, and MpLOX3 proteins were prepared from Escherichia coli cells expressing the corresponding gene. LC–MS/MS analyses and chiral column chromatography of their reaction products showed that MpLOX1 codes for 11S/15S-lipoxygenase against eicosapentaenoic acid and for 15S-lipoxygenase against arachidonic acid, and that MpLOX2 and MpLOX3 code for 15S-lipoxygenase against eicosapentaenoic and arachidonic acids. Phylogenetic analysis showed that the liverwort lipoxygenase genes separated from the ancestor of higher plants in the early stages of plant evolution. Quantification analyses suggested that arachidonic acid and eicosapentaenoic acid were preferred substrates. Furthermore, each liverwort lipoxygenase exhibited highest activity at pH 7.0 and dependency on Ca²⁺ ion in the oxygenation reaction.     

Leukotriene A synthase activity of purified mouse skin arachidonate 8-lipoxygenase expressed in Escherichia coli

Mouse skin 8-lipoxygenase was expressed in COS-7 cells by transient transfection of its cDNA in pEF-BOS carrying an elongation factor-1α promoter. When crude extract of the transfected COS-7 cells was incubated with arachidonic acid, 8-hydroxy-5,9,11,14-eicosatetraenoic acid was produced as assessed by reverse- and straight-phase high performance liquid chromatographies. The recombinant enzyme also reacted on α-linolenic and docosahexaenoic acids at almost the same rate as that with arachidonic acid. Eicosapentaenoic and γ-linolenic acids were also oxygenated at 43% and 56% reaction rates of arachidonic acid, respectively. In contrast, linoleic acid was a poor substrate for this enzyme. The 8-lipoxygenase reaction with these fatty acids proceeded almost linearly for 40 min. The 8-lipoxygenase was also expressed in an Escherichia coli system using pQE-32 carrying six histidine residues at N-terminal of the enzyme. The expressed enzyme was purified over 380-fold giving a specific activity of approximately 0.2 μmol/45 min per mg protein by nickel–nitrilotriacetate affinity chromatography. The enzymatic properties of the purified 8-lipoxygenase were essentially the same as those of the enzyme expressed in COS-7 cells. When the purified 8-lipoxygenase was incubated with 5-hydroperoxy-6,8,11,14-eicosatetraenoic acid, two epimers of 6-trans-leukotriene B₄, degradation products of unstable leukotriene A₄, were observed upon high performance liquid chromatography. Thus, the 8-lipoxygenase catalyzed synthesis of leukotriene A₄ from 5-hydroperoxy fatty acid. Reaction rate of the leukotriene A synthase was approximately 7% of arachidonate 8-lipoxygenation. In contrast to the linear time course of 8-lipoxygenase reaction with arachidonic acid, leukotriene A synthase activity leveled off within 10 min, indicating suicide inactivation.     

Inhibition of mammalian 5-lipoxygenase by aromatic disulfides

As a primary step in leukotriene biosynthesis, arachidonic acid is converted into 5-hydroperoxy-6-trans-8,11,14-cis-eicosatetraenoic acid by 5-lipoxygenase. This enzyme is studied in the supernatant fraction from sonified RBL-1 cells, a preparation that converts [1-14C]arachidonic acid to 5-hydroxy-6-trans-8,11,14-cis-eicosatetraenoic acid and several 5,12-dihydroxyeicosatetraenoic acids including LTB4. In order to examine the reversibility of inhibitors, the supernatant fraction can be depleted of low molecular weight constituents by vacuum filtration. The 5-lipoxygenase is irreversibly inhibited by 500 microM N-ethyl-maleimide or 300 microM methyl methanethiolsulfonate, reagents that react covalently with protein sulfhydryl groups. In contrast, diphenyl disulfide reversibly inhibits this enzyme at 1-5 microM, irrespective of the GSH concentration in the supernatant. KCN also inhibits 5-lipoxygenase at 4 mM, suggesting the presence of a metal-containing prosthetic group. These observations imply that diphenyl disulfide and similar molecules with electron-releasing substituents on the aromatic rings could inhibit by binding to an electrophilic metallic center, the binding being stabilized by hydrophobic interactions between the enzyme and the aromatic groups on the flexible disulfide. Even though diphenyl disulfide does not inhibit soybean 15-lipoxygenase or endoperoxide synthase in cell-free systems, this compound does suppress prostaglandin as well as leukotriene synthesis in intact murine peritoneal macrophages and CXBG cells. Since lipoxygenases are susceptible to peroxide activation and peroxidase deactivation, changes in the redox state of the cell may alter arachidonic acid metabolism as effectively as actual enzyme inhibition.     

Selective feed-back inhibition of the 5-lipoxygenation of arachidonic acid in human T-lymphocytes

Purified human T-lymphocytes exhibit 5-lipoxygenase activity as demonstrated by the conversion of arachidonic acid to 5-hydroxy-eicosatetraenoic acid (5-HETE), 5(S),12(R)-di-hydroxy-eicosa-6,14 cis-8,10 trans-tetraenoic acid (leukotriene B₄), and 5,12-di-HETE isomers of leukotriene B₄ that lack a 6-cis double bond. The concentrations of leukotriene B₄, 5-HETE, 11-HETE and 15-HETE in suspensions of T-lymphocytes were increased significantly by concanavalin A and by the calcium ionophore A23187. Preincubation of T-lymphocytes with 15-HETE at μM concentrations, characteristic of suspensions of stimulated lymphocytes, inhibited selectively the increases in the levels of 5-HETE and leukotriene B₄, but not of 11-HETE and prostaglandin E₂.     

Impact of myeloperoxidase-derived oxidants on the product profile of human 5-lipoxygenase

Human 5-lipoxygenase (5-LOX) oxidizes arachidonic acid to 5S-hydroperoxy-6E,8Z,11Z,14Z-eicosatetraenoic acid (5-HpETE) and leukotriene (LT) A₄. In neutrophils, LTA₄ is further converted to the potent chemoattractant LTB₄. These cells also contain the heme enzyme myeloperoxidase (MPO), which produces several potent oxidants such as hypochlorous acid (HOCl), which are involved in pathogen defense and immune regulation. Here, we addressed the question whether MPO-derived oxidants are able to affect the activity of 5-LOX and the product profile of this enzyme. Human 5-LOX was incubated with increasing amounts of HOCl or HOBr. Afterward, arachidonic acid metabolites of 5-LOX were analyzed by reverse-phase high-performance liquid chromatography as well as by liquid chromatography–electrospray ionization–tandem mass spectrometry. The incubation of 5-LOX with the MPO-derived oxidants significantly changed the product profile of 5-LOX. Thereby, HOCl and HOBr increased the ratio of 5-H(p)ETE to 6-trans-LTB₄ in a concentration-dependent manner. At low oxidant concentrations, there was a strong decrease in the yield of 6-trans-LTB₄, whereas 5-HpETE did not change or increased. Additionally, the formation of 8-HpETE and 12-HpETE by 5-LOX rose slightly with increasing HOCl and HOBr. Comparable results were obtained with the MPO–H₂O₂–Cl⁻ system when glucose oxidase and glucose were applied as a source of H₂O₂. This was necessary because of a strong impairment of 5-LOX activity by H₂O₂. In summary, MPO-derived oxidants showed a considerable impact on 5-LOX, impairing the epoxidation of 5-HpETE, whereas the hydroperoxidation of arachidonic acid was unaffected. Apparently, this was caused by an oxidative modification of critical amino acid residues of 5-LOX. Further work is necessary to assess the specific type and position of oxidation in the substrate-binding cavity of 5-LOX and to specify whether this interaction between 5-LOX and MPO-derived oxidants also takes place in stimulated neutrophils.     

On the Specificity of a Fatty Acid Epoxygenase in Broad Bean (Vicia faba L.)

Seeds of broad bean (Vicia faba L.) contain a hydroperoxide-dependent fatty acid epoxygenase. Hydrogen peroxide served as an effective oxygen donor in the epoxygenase reaction. Fifteen unsaturated fatty acids were incubated with V. faba epoxygenase in the presence of hydrogen peroxide and the epoxy fatty acids produced were identified. Examination of the substrate specificity of the epoxygenase using a series of monounsaturated fatty acids demonstrated that (Z)-fatty acids were rapidly epoxidized into the corresponding cis-epoxy acids, whereas (E)-fatty acids were converted into their trans-epoxides at a very slow rate. In the series of (Z)-monoenoic acids, the double bond position as well as the chain length influenced the rate of epoxidation. The best substrates were found to be palmitoleic, oleic, and myristoleic acids. Steric analysis showed that most of the epoxy acids produced from monounsaturated fatty acids as well as from linoleic and α-linolenic acids had mainly the (R),(S) configuration. Exceptions were C₁₈ acids having the epoxide group located at C-12/13, in which cases the (S),(R) enantiomers dominated. 13(S)-Hydroxy-9(Z),11(E)-octadecadienoic acid incubated with epoxygenase afforded the epoxy alcohol 9(S),10(R)-epoxy-13(S)-hydroxy-11(E)-octadecenoic acid as the major product. Smaller amounts of the diastereomeric epoxy alcohol 9(R),10(S)-epoxy-13(S)-hydroxy-11(E)-octadecenoic acid as well as the α,β-epoxy alcohol 11(R),12(R)-epoxy-13(S)-hydroxy-9(Z)-octadecenoic acid were also obtained. The soluble fraction of homogenate of V. faba seeds contained an epoxide hydrolase activity that catalyzed the conversion of cis-9,10-epoxyoctadecanoic acid into threo-9,10-dihydroxyoctadecanoic acid.     

5-Lipoxygenase-mediated Endogenous DNA Damage

Lipoxygenases (LOs) convert polyunsaturated fatty acids into lipid hydroperoxides. Homolytic decomposition of lipid hydroperoxides gives rise to endogenous genotoxins such as 4-oxo-2(E)-nonenal, which cause the formation of mutagenic DNA adducts. Chiral lipidomics analysis was employed to show that a 5-LO-derived lipid hydroperoxide was responsible for endogenous DNA-adduct formation. The study employed human lymphoblastoid CESS cells, which expressed both 5-LO and the required 5-LO-activating protein (FLAP). The major lipid peroxidation product was 5(S)-hydroperoxy-6,8,11,14-(E,Z,Z,Z)-eicosatetraenoic acid, which was analyzed as its reduction product, 5(S)-hydroxy-6,8,11,14-(E,Z,Z,Z)-eicosatetraenoic acid (5(S)-HETE)). Concentrations of 5(S)-HETE increased from 0.07 ± 0.01 to 45.50 ± 4.05 pmol/10⁷ cells upon stimulation of the CESS cells with calcium ionophore A23187. There was a concomitant increase in the 4-oxo-2(E)-nonenal-derived DNA-adduct, heptanone-etheno-2′-deoxyguanosine (HϵdGuo) from 2.41 ± 0.35 to 6.31 ± 0.73 adducts/10⁷ normal bases. Biosynthesis of prostaglandins, 11(R)-hydroxy-5,8,12,14-(Z,Z,E,Z)-eicosatetraenoic acid, and 15(R,S)-hydroxy-5,8,11,13-(Z,Z,Z,E)-eicosatetraenoic acid revealed that there was cyclooxygenase (COX) activity in the CESS cells. Western blot analysis revealed that COX-1 was expressed by the cells, but there was no COX-2 or 15-LO-1. FLAP inhibitor reduced HϵdGuo-adducts and 5(S)-HETE to basal levels. In contrast, aspirin, which had no effect on 5(S)-HETE, blocked the formation of prostaglandins, 15-HETE, and 11-HETE but did not inhibit HϵdGuo-adduct formation. These data showed that 5-LO was the enzyme responsible for the generation of the HϵdGuo DNA-adduct in CESS cells.PUFAs² can be converted into lipid hydroperoxides enzymatically by the action of LOs (1) and COXs (2) or nonenzymatically by reactive oxygen species (ROS) (3). Arachidonic acid, one of the essential PUFAs present in cell membranes, is released from phospholipids by different phospholipase A₂ isoforms upon diverse physical, chemical, inflammatory, and mitogenic stimuli (4). The free arachidonic acid then serves as a substrate for LOs, COXs, or ROS to produce a variety of lipid hydroperoxides (5, 6). ROS, 12-LO, and 15-LO can also act directly upon arachidonic acid esterified in phospholipids to produce lipid hydroperoxides (7), which are reduced (8), hydrolyzed by phospholipase A₂ (4), and then secreted as the corresponding free HETEs (9). COX-2-mediated (10) and 15-LO-1-mediated (11) metabolism of linoleic acid results in the formation of 13(S)-hydroperoxy-9,11-(Z,E)-octadecadienoic acid, which is rapidly reduced to 13(S)-hydroxy-9,11-(Z,E)-octadecadienoic acid (HODE) and secreted from cells. Arachidonic acid is specifically metabolized by 5-LO into 5(S)-HpETE, which is either reduced to 5(S)-HETE or serves as precursor to the formation of leukotrienes (LTs) (Scheme 1) (12). In contrast, ROS-mediated reactions produce racemic mixtures of all possible regioisomers of HpETEs and 13(S)-hydroperoxy-9,11-(Z,E)-octadecadienoic acids (3) that are subsequently secreted from cells as complex mixtures of racemic HETEs and HODEs. Therefore, the ability to analyze different HETE and HODE enantiomers and regioisomers is important for elucidating specific cellular lipid peroxidation pathways (13).Open in a separate windowSCHEME 1.5-LO-mediated formation of arachidonic acid metabolites and dGuo-adducts. HPNE, 4-hydroperoxy-2(E)-nonenal; DOOE, dioxo-6-octenoic acid.The conversion of arachidonic acid to 5(S)-HpETE by 5-LO is critically dependent upon the presence of FLAP (14). 5-LO and FLAP are expressed primarily in inflammatory cells such as polymorphonuclear leukocytes, monocytes, macrophages, and mast cells (12, 15–17). Therefore, 5-LO-mediated LT formation is thought to play a critical role in inflammation and allergic disorders (18–21). In addition, a number of studies have implicated 5-LO-derived arachidonic acid metabolites as mediators of atherogenesis and heart disease (12, 22, 23). The 5-LO pathway of arachidonic acid metabolism has also been proposed to play a role in prostate and pancreatic cancer (24–26).Lipid hydroperoxides undergo homolytic decomposition into bifunctional electrophiles such as 4-hydroxy-2(E)-nonenal, ONE, 4,5-epoxy-2(E)-decenal, and 4-hydroperoxy-2(E)-nonenal (27). These bifunctional electrophiles are highly reactive and can readily modify intracellular molecules including glutathione (GSH) (28, 29), DNA, (5, 6), and proteins (30, 31). Our previous in vitro studies characterized the bifunctional electrophiles ONE and 4-hydroperoxy-2(E)-nonenal as major products arising from the homolytic decomposition of 5-LO-derived 5(S)-HpETE (32). Reactions with DNA resulted in the formation of etheno-2′-deoxyguanosine (ϵdGuo) from 4-hydroperoxy-2(E)-nonenal and heptanone-ϵdGuo (HϵdGuo) from ONE (Scheme 1). 5,8-Dioxo-6-octenoic acid, a bifunctional electrophile from the carboxyl terminus of 5(S)-HpETE, gave rise to the novel DNA-adduct carboxypentanone-ϵdGuo (CPϵdGuo)-adduct as shown in Scheme 1.Previous studies have demonstrated that lipid hydroperoxides generated by COX-2 could lead to the formation of endogenous DNA adducts in epithelial cells (6). Cellular 5-LO, like COX-2, synthesizes lipid hydroperoxides on the nuclear membrane. Therefore, it is highly possible that 5-LO could also mediate the formation of lipid hydroperoxide-derived endogenous DNA adducts in cells. CESS is a human lymphoblastic cell line that expresses both 5-LO and FLAP, and they have been used as a model for inflammatory cells to examine the role of 5-LO metabolites in signal transduction (33, 34). In the present study, CESS cells provided an ideal model to elucidate the relationship of 5-LO mediated-lipid peroxidation and DNA-adduct formation in a cellular setting. Stable isotope dilution chiral LC-electron capture (EC) APCI/MRM/MS (13) was used to monitor the concomitant formation of lipid hydroperoxides in the presence of different enzyme stimulator or inhibitors. DNA-adduct formation in the same cells was measured by a stable isotope dilution APCI/MRM/MS method. The powerful tool of chiral lipid analysis enabled us to dissect the complicated lipid peroxidation pathways and to correlate them with endogenous DNA-adduct formation. The results demonstrated that 5-LO-mediated lipid peroxidation could cause HϵdGuo formation in cells. This novel finding provided additional explanation for the previous observation that increased 5-LO activity was associated with cancers and cardiovascular diseases (24–26).     

Hydroxy-alkenals from the peroxidation of n-3 and n-6 fatty acids and urinary metabolites

4-Hydroxy-2E-hexenal (4-HHE) and 4-hydroxy-2E-nonenal (4-HNE) have been characterized as prominent by-products of n-3 and n-6 hydroperoxy derivatives of n-3 and n-6 fatty acids, respectively. We also have characterized the homolog 4-hydroxy-2E,6Z-dodecadienal (4-HDDE) as a specific by-product of the 12-lipoxygenase product of arachidonic acid 12-hydroperoxy-eicosatetraenoate (12-HpETE). The three hydroxy-alkenals have been found in human plasma with 4-HHE being the most prominent followed by 4-HNE. They were found increased in tissues submitted to oxidative stress, according to the fatty acid characteristic of those tissues, e.g., 4-HNE and 4-HDDE in blood platelets and 4-HHE in the retina. We have shown they covalently bind to the primary amine moiety of ethanolamine phospholipids (PE), especially the plasmalogen subclass, with the highest hydrophobic alkenal (4-HDDE) being the most reactive. Their carboxylic acid metabolites, 4-hydroxy-2E-hexenoic acid (4-HHA), 4-hydroxy-2E-nonenoic acid (4-HNA) and 4-hydroxy-2E,6Z-dodecadienoic acid (4-HDDA), respectively, were found in human urine and measured in higher amounts in situations in which oxidative stress has been reported such as aging and diabetes. As reported above with their hydroxy-alkenals precursors, 4-HHA proved to be the most prominent followed by 4-HNA. Altogether, the three hydroxy-alkenals, either in their free form or bound to membrane PE, may be considered as specific markers of lipid peroxidation able to discriminate between n-3 and n-6 fatty acids. This is corroborated by the measurement of their urinary carboxylic acid metabolites.     

Contributory Role of BLT2 in the Production of Proinflammatory Cytokines in Cecal Ligation and Puncture-Induced Sepsis

BLT2 is a low-affinity receptor for leukotriene B₄, a potent lipid mediator of inflammation generated from arachidonic acid via the 5-lipoxygenase pathway. The aim of this study was to investigate whether BLT2 plays any role in sepsis, a systemic inflammatory response syndrome caused by infection. A murine model of cecal ligation and puncture (CLP)-induced sepsis was used to evaluate the role of BLT2 in septic inflammation. In the present study, we observed that the levels of ligands for BLT2 (LTB₄ [leukotriene B₄] and 12(S)-HETE [12(S)-hydroxyeicosatetraenoic acid]) were significantly increased in the peritoneal lavage fluid and serum from mice with CLP-induced sepsis. We also observed that the levels of BLT2 as well as 5-lipoxygenase (5-LO) and 12-LO, which are synthesizing enzymes for LTB₄ and 12(S)-HETE, were significantly increased in lung and liver tissues in the CLP mouse model. Blockade of BLT2 markedly suppressed the production of sepsis-associated cytokines (IL-6 [interleukin-6], TNF-α[[tumor necrosis factor alpha], and IL-1β [interleukin-1β] as well as IL-17 [interleukin-17]) and alleviated lung inflammation in the CLP group. Taken together, our results suggest that BLT2 cascade contributes to lung inflammation in CLP-induced sepsis by mediating the production of inflammatory cytokines. These findings suggest that BLT2 may be a potential therapeutic target for sepsis patients.     

On the reaction specificity of the lipoxygenase from tomato fruits

A lipoxygenase was purified 300-fold from a homogenate supernatant of ripe tomato fruits by fractionated ammonium sulfate precipitation and anion exchange fast protein liquid chromatography. The specific linoleate oxygenase activity of the final enzyme preparation was 1300 nkat per mg protein at pH 6.8 and 25°C in the absence of any detergent. The enzyme oxygenated linoleic acid and α-linolenic acid at comparable rates, whereas γ-linolenic acid, arachidonic acid, 11,14-eicosadienoic acid and 11,14,17-eicosatrienoic acid were poor substrates. Linoleic acid was converted to 9(S)-hydroperoxy-10E,12Z-octadecadienoic acid, whereas 5(S)-HpETE, 11(S)-HpETE and 8(S)-HpETE were identified as major oxygenation products from arachidonic acid. The tomato lipoxygenase did not react with either dilinoleyl phosphatidylcholine or the lipid extract from beef heart mitochondria. The possible biological importance of the reaction of tomato lipoxygenase with arachidonic acid is discussed.     

Evidence for in situ ethanolamine phospholipid adducts with hydroxy-alkenals

Hydroxy-alkenals, such as 4-hydroxy-2(E)-nonenal (4-HNE; from n-6 fatty acids), are degradation products of fatty acid hydroperoxides, including those generated by free radical attack of membrane polyunsaturated fatty acyl moieties. The cytotoxic effects of hydroxy-alkenals are well known and are mainly attributable to their interaction with different molecules to form covalent adducts. Indeed, ethanolamine phospholipids (PEs) can be covalently modified in a cellular system by hydroxy-alkenals, such as 4-HNE, 4-hydroxy-2(E)-hexenal (4-HHE; from n-3 fatty acids), and 4-hydroxy-dodecadienal (4-HDDE; from the 12-lipoxygenase product of arachidonic acid), to form mainly Michael adducts. In this study, we describe the formation of PE Michael adducts in human blood platelets in response to oxidative stress and in retinas of streptozotocin-induced diabetic rats. We have successfully characterized and evaluated, for the first time, PEs coupled with 4-HHE, 4-HNE, and 4-HDDE by gas chromatography-mass spectrometry measurement of their ethanolamine moieties. We also report that aggregation of isolated human blood platelets enriched with PE-4-hydroxy-alkenal Michael adducts was altered. These data suggest that these adducts could be used as specific markers of membrane disorders occurring in pathophysiological states with associated oxidative stress and might affect cell function.     

A 4-oxo-2(E)-nonenal-derived glutathione adduct from 15-lipoxygenase-1-mediated oxidation of cytosolic and esterified arachidonic acid

15(S)-Hydroperoxy-[5Z,8Z,11Z,13E]-eicosatetraenoic acid (15(S)-HpETE) undergoes homolytic decomposition to bifunctional electrophiles such as 4-oxo-2(E)-nonenal. 4-Oxo-2(E)-nonenal reacts with glutathione to form a thiadiazabicyclo-4-oxo-2(E)-nonenal–glutathione adduct (TOG). Therefore, this endogenous glutathione adduct can serve as a specific biomarker of lipid hydroperoxide-mediated 4-oxo-2(E)-nonenal formation. A monocyte/macrophage cell line was generated to constitutively express human 15-lipoxygenase-1. In these cells, TOG was formed from 15(S)-HpETE-derived 4-oxo-2(E)-nonenal in a nonlinear dose-dependent manner upon arachidonic acid treatment. The lipoxygenase inhibitor cinnamyl-3,4-dihydroxy-α-cyanocinnamate abolished arachidonic acid-mediated TOG formation. The calcium ionophore A23187 was also used to induce the formation of 15(S)-HpETE from esterified arachidonic acid present in the membrane lipids. In the 15-lipoxygenase-1-expressing cells, the calcium ionophore A23187 significantly increased TOG levels compared with mock-transfected cells. This was due to the 15-lipoxygenase-mediated formation of 15(S)-HpETE in the forms of free fatty acid and esterified lipids, which was subsequently converted to 4-oxo-2(E)-nonenal. The increase in TOG formation was again abrogated by pretreatment with cinnamyl-3,4-dihydroxy-α-cyanocinnamate. Only 8.7% 15(S)-HETE (both the free fatty acid and its esterified form in the cell membrane) was formed after ionophore A23187 stimulation compared with that formed after the addition of arachidonic acid. In contrast, the TOG levels after treatment with ionophore A23187 or arachidonic acid were comparable. Thus, it is likely that esterified 15(S)-HpETE underwent homolytic decomposition to 4-oxo-2(E)-nonenal more efficiently than the free 15(S)-HpETE that was formed in the cytosol.     

Stereoselective hydrogen abstraction in leukotriene A4 synthesis by purified 5-lipoxygenase of porcine leukocytes

Arachidonate 5-lipoxygenase purified from porcine leukocytes transformed arachidonic acid to 5-hydroperoxy-6,8,11,14-eicosatetraenoic acid. By the leukotriene A synthase activity of the same enzyme the product was further metabolized to leukotriene A₄ (actually detected as 6-trans-leukotriene B₄, 12-epi-6-trans-leukotriene B₄, abd 5,6-duhydroxy-7,9,11,14-eicosatetraenoic acids). The enzyme was incubated with [10-D_R-³H]- or [10-L_S-³H]- labeled arachidonic acid, and 6-trans-LTB₄ and its 12-epimer were analyzed. More than 90% of 10-D_R-hydrogen was lost while about 100% of 10-L_S-hydrogen was retained, indicating a stereospecific hydrogen elimination from C-10 during the formation of leukotriene A₄.     

Concerning the biosynthesis of prostaglandin I2

Two diastereoisomers, 5R,6R-5-hydroxy-6(9α)-oxido-11α,15S-dihydroxyprost-13-enoic acid (7) and 5S,6S-5-hydroxy-6(9α)-oxido-11α,15S-dihydroxyprost-13-enoic acid (10) were synthesized for evaluation as possible biosynthetic intermediates in the enzymatic transformation of PGH₂ or PGG₂ into PGI₂. The synthetic sequence entails the stereospecific reduction of the 9-keto function in PGE₂ methyl ester after protecting the C-11 and C-15 hydroxyls as tbutyldimethylsilyl ethers. The resulting PGF_2α derivative was epoxidized exclusively at the C-5 (6) double bond to yield a mixture of epoxides, which underwent facile rearrangement with SiO₂ to yield the 5S,6S and 5R,6R-5-hydroxy-6(9α)-oxido cyclic ethers. It was found that dog aortic microsomes were unable to transform radioactive 9β-5S,6S[³H] or 9β-5R,6R[³H]-5-hydroxy-6(9α)-oxido cyclic ethers into PGI₂. Also, when either diastereoisomer was included in the incubation mixture, neither isomer diluted the conversion of [1-¹⁴C]arachidonic acid into [1-¹⁴C]PGI₂.     

Oxidation of the primary hydroxyl group of galactose of galactaosyl ceramide analogue by chemical method—precursors for the synthesis of labeled conjugates

Isotopic labeling of the C-6 of a model glycosphingolipid (2S, 3R, 4E)-2-(1-adamantanacetamido)-3-hydroxy-4-octadecenyl-β-d-galactopyranoside, GalCAda, is described. Oxidation of (2S, 3R, 4E)-2-(1-adamantanacetamido)-3-(benzoyloxy)-4-octadecenyl-2,3,4-tri-O-benzoyl-β-d-galactopyranoside with o-iodoxybenzoic acid gave the dialdoside derivative in good yield. Reduction of the dialdoside with sodium borodeuteride gave the deuterium labeled d-GalCAda, with a cumulative yield of 35%.