Novel leukotrienes: Products formed by initial oxygenation of arachidonic acid at C-15

A preparation of human leukocytes was incubated with arachidonic acid. Two new dihydroxy acids with conjugated triene structures, were isolated and characterized as 8,15-dihydroxy-5,9,11,13-eicosatetraenoic acid (8,15-leukotriene B₄) and 14,15-dihydroxy-5,8,10,12-eicosatetraenoic acid (14,15-leukotriene B₄).     

Leukotriene formation by a purified reticulocyte lipoxygenase enzyme. Conversion of arachidonic acid and 15-hydroperoxyeicosatetraenoic acid to 14, 15-leukotriene A4

The purified lipoxygenase of rabbit reticulocytes converts arachidonic acid at 0 degrees C to 15-hydroperoxyeicosatetraenoic acid (15-HPETE) and to 12-hydroperoxyeicosatetraenoic acid (12-HPETE) via reactions which involve hydrogen abstraction at C-13 and C-10, respectively. At 37 degrees C the enzyme converts arachidonic acid to additional products which were identified as 13-hydroxy-14,15-epoxy-5,8,11-eicosatrienoic acid, 8,15-dihydroperoxy-5,9,11,13- and 5,15-dihydroperoxy-6, 6,8,11,13-eicosatetraenoic acids (8,15-diHPETE and 5,15-HPETE, respectively) and diastereoisomers of 8,15-dihydroxy-5,9,11,13-eicosatetraenoic acid (8,15-diHPETEs). The 8,15- and 5,15-diHPETEs were formed by double lipoxygenation since each incorporated 2 molecules of 18O2 and since their synthesis from 15-HPETE was blocked under anaerobic conditions. The 8,15-diHETEs each incorporated 18O from 18O2 at C-15 and were found to arise from nonenzymatic hydrolysis of an epoxytriene which was identified as 14,15-leukotriene A4 by trapping in acidic methanol. This compound was a major product of 15-HPETE in anaerobic incubations. The conversion of 15-HPETE to 14,15-leukotriene A4 was inhibited by the lipoxygenase inhibitors nordihydroguairetic acid and 5,8,11,14-eicosatetraynoic acid. The 14,15-leukotriene A4 synthase and 15-lipoxygenase activities were inhibited by 5,8,11,14-eicosatetraynoic acid in a similar time-dependent manner. The results support a mechanism whereby 14,15-leukotriene A4 is synthesized from 15-HPETE by a further enzymatic step carried out by the reticulocyte 15-lipoxygenase via hydrogen abstraction at C-10 and a redox cycle of the non-heme iron atom of the enzyme.     

Hemoprotein catalysis of leukotriene formation

Incubation of various hemoproteins with 5-hydroperoxy-6,8,11,14-eicosatetraenoic acid or 15-hydroperoxy-5,8,11,13-eicosatetraenoic acid resulted in formation of epimeric 5(S),12-dihydroxy-6,8,10,14 -eicosatetraenoic acids and epimeric 8,15(S)-dihydroxy-5,9,11,13 -eicosatetraenoic acids, respectively. These dihydroxy acids were earlier recognized as nonenzymatic hydrolysis products of 5(S),6-oxido-7,9,11,14-eicosatetraenoic acid (leukotriene A4) and 14,15(S)-oxido-5,8,10,12-eicosatetraenoic acid (14,15-leukotriene A4). These allylic epoxides could be isolated as such from the hemoprotein incubations, and most probably they are intermediates in formation of the dihydroxy acids.     

Biosynthesis and metabolism of 15-hydroperoxy-5,8,11,13-eicosatetraenoic acid by human umbilical vein endothelial cells

Incubation of cultured human umbilical vein endothelial cells with [1-14C]arachidonic acid, followed by reverse-phase high-pressure liquid chromatography analysis, results in the appearance of two principal radioactive products besides 6-keto-prostaglandin F1 alpha. The first peak is 12-L-hydroxy-5,8,10-heptadecatrienoic acid, a hydrolysis product of the prostaglandin endoperoxide. The second peak was esterified, converted to the trimethylsilyl ether derivative, and analyzed by gas chromatography-mass spectrometry and shown to be the lipoxygenase product 15(S)-hydroxy-5,8,11,13-eicosatetraenoic acid (15-HETE). Incubation of the 15-HETE precursor 15(S)-hydroperoxy-5,8,11,13-eicosatetraenoic acid (15-HPETE) with endothelial cells results in the formation of four distinct UV absorbing peaks. UV and gas chromatography-mass spectrometry analysis showed these peaks to be 8,15(S)-dihydroxy-5,8,11,13-eicosatetraenoic acids (8,15-diHETE) differing only in their hydroxyl configuration and cis trans double-bond geometry. Formation of 8,15-diHETE molecules suggests the prior formation of the unstable epoxide molecule 14(S),15(S)-trans-oxido-5,8-Z-14,15-leukotriene A4 or an attack at C-10 of 15-HPETE by an enzyme with mechanistic features in common with a 12-lipoxygenase. The observation that endothelial cells can synthesize both 15-HETE and 8,15-diHETE molecules suggests that this cell type contains both a 15-lipoxygenase and a system that can synthesize 14,15-leukotriene A4.     

Stereochemistry, total synthesis, and biological activity of 14,15-dihydroxy-5,8,10,12-eicosatetraenoic acid

The stereochemistry of the major isomer of 14,15-dihydroxy-5,8,10,12-eicosatetraenoic acid formed from 15-hydroperoxyeicosatetraenoic acid in human leukocytes was determined. The structure (erythro-14(R),15(S]-14,15-dihydroxy-5,8-cis-10,12-trans-eicosatetraenoi c acid) was assigned based on sodium arsenite thin-layer chromatography, NMR spectroscopy, and comparison with material prepared by total synthesis. This compound was found to inhibit leukotriene B4-induced superoxide anion generation in human neutrophils (IC50 = 10(-8)-10(-7) M). Superoxide anion generation induced by either formylmethionyl-leucyl-phenylalanine or arachidonic acid was not affected.     

Metabolism of arachidonic acid by human nasal and bronchial epithelial cells

Nasal and bronchial epithelium from normal human nasal turbinates was isolated from surgical specimens and used to study arachidonic acid metabolism. High-performance liquid chromatography analysis of cell incubations in the presence of calcium ionophore, A23187, showed the formation of 15-lipoxygenase products. The major arachidonic acid metabolite with bronchial and nasal tissue was 15-HETE identified by uv spectroscopy, coelution with the authentic standards by HPLC, and GC-mass spectrometry. The second major metabolite, formed from either arachidonic acid or 15-HPETE, was identified as 13-hydroxy-14,15-epoxy-5,8,11-eicosatetraenoic acid (15-alpha-HEPA) by uv spectroscopy, coelution with the authentic standard, and GC-mass spectrometry. In addition, two 8,15-diHETEs and two 8,15-LTs were identified by uv spectroscopy and coelution with the authentic standards by HPLC on both reverse-phase and normal-phase HPLC. Also isolated and identified were 14,15-diHETEs, and 12-HETE. Nasal epithelial cells appear to be more active than nasal bronchial cells in oxidizing arachidonic acid. However, the profile of metabolites from these normal tissue preparations was similar. The addition of 15-lipoxygenase products to nasal epithelium weakly stimulated Cl- ion secretion. These studies indicate that human pulmonary epithelial cells selectively oxidize arachidonic acid to 15-lipoxygenase metabolites.     

Calcium-dependent oxidation of 5,8,11-icosatrienoic acid by the cyclooxygenase enzyme system

Mead (5,8,11-icosatrienoic) acid was found to be metabolized by the cyclooxygenase enzyme system of ram seminal vesicle microsomes in a calcium-dependent manner. Although the enzyme converted Mead acid to products more slowly and less completely than the isomeric 8,11,14-icosatrienoic acid, both oxidations were inhibitable by indomethacin. Experiments using purified cyclooxygenase confirmed the participation of this enzyme system in the calcium-dependent oxidation. The products of the oxidation were separated by high performance liquid chromatography and analyzed by ultraviolet and gas chromatography-mass spectrometry. The spectra obtained were consistent with the products having the structures 13-hydroxy-5,8,11-icosatrienoate (the major product), 11-hydroxy-5,8,12-icosatrienoate, 9-hydroxy-5,7,11-icosatrienoate, and two isomeric 8,11-dihydroxy-5,9,12-icosatrienoates. No prostaglandin-like, cyclized products could be identified. This report is only the second to illustrate a calcium-dependent oxidation of a polyunsaturated fatty acid by a cyclooxygenase enzyme system and further extends the metabolic potential of Mead acid.     

A novel dioxygenation product of arachidonic acid possesses potent chemotactic activity for human polymorphonuclear leukocytes

We have found that a novel dioxygenation product of arachidonic acid, 8(S),15(S)-dihydroxy-5,11-cis-9,13-trans-eicosatetraenoic acid (8,15-diHETE), possesses chemotactic activity for human polymorphonuclear leukocytes comparable to that of leukotriene B4. Authentic 8,15-diHETE, identified by gas chromatography-mass spectrometry, was prepared by treating arachidonic acid with soybean lipoxygenase and was purified by reverse-phase high performance liquid chromatography. Using a "leading front" assay, 8,15-diHETE exhibited significant chemotactic activity at a concentration of 5.0 ng/ml. Maximum chemotactic activity was observed at a concentration of 30 ng/ml. The 8,15-diHETE generated by mixed human leukocytes after stimulation with arachidonic acid and the calcium ionophore, A23187, exhibited quantitatively similar chemotactic activity. Two synthetic all-trans conjugated isomers of 8,15-diHETE, however, were not chemotactic at concentrations up to 500 ng/ml. In contrast to its potent chemotactic activity, 8,15-diHETE (at concentrations up to 10 micrograms/ml) was relatively inactive with respect to its ability to provoke either degranulation or generation of superoxide anion radicals by cytochalasin B-treated leukocytes. Both leukotriene B4 and 8,15-diHETE may be important mediators of inflammation.     

New natural diterpene acids from Juniperus communis

Three new diterpene acids have been isolated from the leaves of Juniperus communis and their structures, elucidated by spectroscopic methods, were identified as 7-oxo-13-epi- pimara-8,15-dien-18-oic acid, 7α-hydroxysandaracopimaric acid and (14 S)-14,15-dihydroxylabda- 8(17),13(16)-dien-19-oic acid. Biflavonyls, fatty acids and diterpenoids with known structures were also isolated.     

14,15-Dihydroxy-5,8,10,12-eicosatetraenoic acid. Enzymatic formation from 14,15-leukotriene A4

When 14C-labeled (14S, 15S)-14,15-trans-oxido-5,8-cis-10,12-trans-eicosatetraenoic acid (14,15-leukotriene A4) was incubated with cytosolic epoxide hydrolase purified from mouse liver, one major radiolabeled product appeared. The structure was assigned as (14R, 15S)-14,15-dihydroxy-5,8-cis-10,12-trans-eicosatetraenoic acid (14,15-DHETE), based on analytical data as well as enzyme mechanistic considerations. The formation of this compound was dependent on time and enzyme concentration and was abolished after heat treatment of the enzyme. The apparent Km and Vmax values at 37 degrees C were 11 microM and 900 nmol X mg-1 X min-1 respectively. This enzymatic hydrolysis of 14,15-leukotriene A4 represents an additional mode of formation for 14,15-DHETE, a compound previously found to modulate functions of human leukocytes.     

Transformation of 15-hydroperoxyeicosapentaenoic acid to lipoxin A5 and B5, mono- and dihydroxyeicosapentaenoic acids by porcine leukocytes

15-Hydroperoxy[1-14C]eicosapentaenoic acid derived from eicosapentaenoic acid (EPA) was incubated with suspensions of porcine leukocytes. Incubation with porcine leukocytes resulted in the formation of seven dihydroxy compounds, one monohydroxy and one hydroxyepoxy compound. After separation by reverse-phase and straight-phase HPLC, GC/MS analysis revealed that these metabolites were four isomers of 8,15-diHEPEs, two isomers of 14,15-diHEPEs, one isomer of 5,15-diHEPE, 15-HEPE and an epoxyalcohol: 13-hydroxy-14,15-epoxyeicosatetraenoic acid. In addition to the above metabolites, two trihydroxytetraene derivatives were also isolated. GC/MS and ultraviolet spectroscopy identified the two trihydroxypentaene derivatives as 5,6,15-trihydroxy-7,9,11,13,17-eicosapentaenoic acid (lipoxin A5) and 5,14,15-trihydroxy-6,8,10,12,17-eicosapentaenoic acid (lipoxin B5). This study demonstrated that the 15-hydroperoxide of EPA can be actively converted to various hydroxylated products via the 5-, 12- and 15-lipoxygenase as well as epoxyisomerase pathways in the porcine leukocytes.     

Ceriopsins F and G,diterpenoids from Ceriops decandra

Chemical examination of the ethyl acetate solubles of the CH(3)OH:CH(2)Cl(2) (1:1) extract of the roots of Ceriops decandra collected from Kauvery estuary resulted in the isolation of two more diterpenoids, ceriopsins F and G (1-2) and five known compounds, ent-13-hydroxy-16-kauren-19-oic acid (steviol, 3), methyl ent-16beta,17-dihydroxy-9(11)-kauren-19-oate (4), ent-16beta,17-dihydroxy-9(11)-kauren-19-oic acid (5), ent-16-oxobeyeran-19-oic acid (isosteviol, 6), 8,15R-epoxypimaran-16-ol (7). The structures of the new diterpenoids were elucidated by a study of their physical and spectral data as methyl ent-13,17-epoxy-16-hydroxykauran-19-oate (1) and ent-16-oxobeyeran-19-al (2).     

Rabbit renal cortical microsomes metabolize arachidonic acid to trihydroxyeicosatrienoic acids

(1-¹⁴C) Eicosatetraenoic (Arachidonic) acid was incubated wiht microsomes from rabbit renal cortex and NADPH (1 mM) for 15 min at 37°C. The products were extracted and purified by high pressure liquid chromatography. Some of the most polar metabolites were identified by gas chromatography mass spectrometry. They were 11, 12, 19- and 11, 12,20-trihydroxy-5,8-14-eicosatrienoic acid, 14,15,19- and 14,15,20- trihydroxy-5,8,11-eicosatrienoic acid, and 11,12-dihydroxy-19-oxo- 5,8,14-eicosatrienoic acid. These products were likely formed by ω- and (ω−1)-hydroxylation of 11,12-dihydroxy-5,8,14-eicosatrienoic aic and 14,15-dihydroxy-5,8,11-eicosatrienoic acid, two recently identified metabolites of arachidonic acid in fortified rabbit kidney microsomes.     

Mechanistic studies of the dioxygenase and leukotriene synthase activities of the porcine leukocyte 12S-lipoxygenase

Lipoxygenases react with hydroperoxy fatty acids and catalyze dioxygenase or dehydrase (leukotriene A4 (LTA4) synthase) types of reactions. In the present investigation we studied the mechanism of reaction of the purified porcine leukocyte 12S-lipoxygenase with 15S-hydroperoxyeicosatetraenoic acid (15S-HPETE). Oxygen-18 labeling experiments with GC-MS analysis were used to distinguish dioxygenase and leukotriene synthase activities of the enzyme; 8S,15S-DiHPETE and 14R,15S-DiHPETE were formed by oxygenation, and a series of 8,15- and 14,15-diols were formed via enzymatic synthesis of 14,15-LTA4 and nonenzymatic hydrolysis of the epoxide. 10D-3H- and 10L-3H-labeled substrates were used to study the stereospecificity of the C-10 hydrogen abstraction in the synthesis of these products. Formation of 14,15-DiHPETE and 14,15-LTA4 was associated with stereoselective abstraction of hydrogen from the 10L position of 15S-HPETE. The same type of measurements on the 8S,15S-DiHPETE product indicated a variable (50-250%) retention of the 10L-3H label, and a consistent 90% retention of the 10D-3H. In contrast, the synthesis of 8S,15S-DiHPETE by the soybean lipoxygenase was associated with the expected stereoselective abstraction of the 10D hydrogen. It appears that the porcine leukocyte 12S-lipoxygenase synthesizes 8S,15S-DiHPETE by a different mechanism.     

Metabolism of arachidonic acid by isolated rat hepatocytes,renal cells and by some rabbit tissues: Detection of vicinal diols by mass fragmentography

Purified cytochromes P-450 (LM₂ and PB-B₂) in a reconstituted system and epoxide hydrolase were recently found to metabolize arachidonic (eicosatetraenoic) acid to four vicinal dihydroxyeicosatrienoic acids. These metabolites were chemically synthetized from octadeuterated arachidonic acid and employed as internal standards for mass fragmentography. Isolated rat hepatocytes and renal cells were incubated with arachidonic acid (0.1 mM; 37°C, 15 min) and, following extractive isolation and reversed-phase HPLC, formation of 11,12-dihydroxy-5,8,14-eicosatrienoic acid and 14,15-dihydroxy-5,8,11-eicosatrienoic acid was demonstrated by mass fragmentography using a capillary GC column. Furthermore, these diols were also detected in rabbit liver and renal cortex and they therefore appear to be formed endogenously. Formation of vicinal diols was also studied in cell free systems. Rabbit liver and renal cortical microsomes were incubated with NADPH (1 mM) and arachidonic acid (0.15 mM) for 15 min at 37°C and, besides 11,12-dihydroxy- and 14,15-dihydroxyeicosatrienoic acid, small amounts of 8,9-dihydroxy- and 5,6-dihydroxyeicosatrienoic acid could be detected by mass fragmentography. Renal as well as hepatic monooxygenases can thus epoxidize each of the four double bonds of arachidonic acid. In contrast, rabbit lung microsomes and NADPH metabolize arachidonic acid mainly to prostaglandins and 19-hydroxy- and 20-hydroxyarachidonic acid, while only small amounts of 11,12-dihydroxyeicosatrienoic acid could be found. Monooxygenase metabolism of arachidonic acid by epoxidation might therefore be a significant pathway for the metabolism of this essential fatty acid in isolated rat renal cells and hepatocytes but presumably not in the lung.     

Evidence for a novel pathway of leukotriene formation in human platelets

The metabolism of arachidonic acid via lipoxygenase-catalyzed reactions in washed human platelets was investigated. In addition to the previously discovered lipoxygenase metabolites, 12-hydroxyeicosatetraenoic acid, 15-hydroxyeicosatetraenoic acid, 8,15-dihydroxyeicosatetraenoic acid and 14,15-dihydroxyeicosatetraenoic acid, several other products were formed. The compounds were all dihydroxylated metabolites of arachidonic acid, containing a conjugated triene structure, and identified as 11,12-dihydroxyeicosatetraenoic acid (two isomers) and 5,12-dihydroxyeicosatetraenoic acid (four isomers). The identification was based on ultraviolet spectroscopy and gas chromatography-mass spectrometry of native and hydrogenated compounds. Stereochemical analysis of the hydroxyl groups of the 5,12-dihydroxyeicosatetraenoic acids and experiments with 18O2 indicated that the compounds were formed by the 12-lipoxygenase pathway, probably via an unstable epoxide.     

Role of cytosolic free calcium and phospholipase C in leukotriene-B4-stimulated secretion in human neutrophils. Comparison with the chemotactic peptide formyl-methionyl-leucyl-phenylalanine

Various leukotriene analogues were tested for their capacity to raise the cytosolic free calcium concentration, [Ca2+]i, and to stimulate exocytosis in human neutrophils. Their order of potency for both parameters was LTB4 greater than the stereochemical isomer of LTB4, (5S, 12S)-LTB4 much much greater than the sulphidopeptides LTD4, LTC4. The correlation between [Ca2+]i and secretion indicates that an increase of [Ca2+]i above a threshold level of about 300 nM is necessary for stimulating secretion with LTB4. This threshold is about an order of magnitude higher than that required for the chemotactic peptide formyl-methionyl-leucyl-phenylalanine (fMet-Leu-Phe). The increase in [Ca2+]i elicited by LTB4 was unaffected by increasing cellular cAMP, while secretion was completely inhibited. These results indicate, that similar to fMet-Leu-Phe, leukotrienes generate other signals in addition to [Ca2+]i elevations. Contrary to previous claims, leukotrienes stimulate polyphosphoinositide hydrolysis, as indicated by the increase in [3H]inositol trisphosphate, InsP3, observed upon stimulation of myo[3H]inositol-labelled neutrophils with LTB4 or (5S, 12S)-LTB4. The two InsP3 isomers [Ins(1,4,5)P3 and Ins(1,3,4P3] were separated by high-pressure liquid chromatographed and, as reported for other cell types, the formation of Ins(1,4,5)P3 precedes that of Ins(1,3,4)P3. Maximal stimulatory doses of LTB4 or (5S, 12S)-LTB4 produce about 50% the amount of InsP3 generated by equimolar concentrations of fMet-Leu-Phe. The present observations suggest that, though the transmembrane signalling systems activated by LTB4 and fMet-Leu-Phe are the same, the different efficacy of these two agonists at stimulating neutrophil functions is due, at least in part, to a different degree of activation of phospholipase C.     

Mechanisms of leukotriene formation: hemoglobin-catalyzed transformation of 15-HPETE into 8,15-DiHETE and 14,15-DiHETE isomers

Four isomers of 8,15-diHETE as well as 14,15-diHETEs are isolated and characterized after exposure of 15-HPETE to hemoglobin. It is found that 83% of the C-8 oxygen atoms in 8(R), 15(S)-diHETE and 8(S), 15(S)-diHETE, and 41% of the C-8 oxygen atoms in 8(R), 15(S)-11Z-diHETE and 8(S), 15(S)-11Z-diHETE are derived from H2(18)O. These results suggest that hemoglobin catalyzes the transformation of 15-HPETE into these products via a free radical process, possibly involving the intermediacy of 14,15-LTA. Intact human leukocytes contain a distinct enzyme system for catalyzing the conversion of 15-HPETE into 14,15-LTA. This enzyme activity is inhibited by ETYA and is rapidly denatured upon homogenization of the intact leukocytes.     

Appearance of an arachidonic acid 15-lipoxygenase pathway upon differentiation of the human promyelocytic cell-line HL-60

The metabolism of arachidonic acid and 15-HPETE was studied in a human promyelocytic cell line (HL-60). Upon exposure to DMSO, HL-60 cells undergo differentiation and acquire a 15-lipoxygenase activity while undifferentiated cells challenged with either arachidonic acid or 15-HPETE did not enzymatically transform these precursors. Products of the arachidonic acid 15-lipoxygenase pathway were identified by HPLC. UV-absorption and gas chromatography-mass spectrometry. Results indicate that upon differentiation HL-60 cells express a 15-lipoxygenase activity as well as the ability to transform 15-HPETE to 8,15-DHETEs and 14,15-DHETE. Moreover, these findings suggest that products of the 15-lipoxygenase cascade may be generated by a single cell system.     

Arachidonic acid metabolism in rabbit renal cortex. Formation of two novel dihydroxyeicosatrienoic acids

[1-14C]Eicosatetraenoic (arachidonic) acid was incubated with a low speed (17,000 X g) rabbit renal cortical supernatant or with a cortical microsomal suspension fortified with NADPH for 15 min at 37 degrees C. The products which were less polar than prostaglandins on reversed phase high performance liquid chromatography were identified by gas chromatography-mass spectrometry. Both the fortified microsomes and the low speed supernatant formed significant amounts of two novel metabolites, 11,12-dihydroxy-5,8,14-eicosatrienoic acid and 14,15-dihydroxy-5,8,11-eicosatrienoic acid. Other identified products were 19- and 20-hydroxyeicosatetraenoic acid, 19-oxoeicosatetraenoic acid, and in the low speed supernatant, eicosatetraen-1,20-dioic acid. The metabolites were not formed in significant amounts by high speed cortical supernatant or by nonfortified cortical microsomes. Carbon monoxide inhibited formation of these compounds, indicating that they may be formed by the cytochrome P-450-linked renal monooxygenase systems.