Biomedical subjects
J Rokach
Publications and source records attributed to J Rokach.
Synthesis of beta-oxidation products as potential leukotriene metabolites and their detection in bile of anesthetized rat.
Two novel beta-oxidation products of peptido leukotrienes, 16-carboxy-17,18,19,20-tetranor-14,15-dihydro-N-acetyl LTE4 and 18-carboxy-19,20-dinor-N-acetyl LTE4, were prepared by total synthesis and used to identify previously unknown polar rat biliary metabolites. When [3H] LTC4 and synthetic N-acetyl-LTE4 were administered intravenously to anesthetized inbred male rats, extraction of the bile and subsequent reverse-phase HPLC fractionation allowed the isolation of two novel metabolites of N-acetyl-LTE4. Comparison of U.V. spectra and coelution experiments revealed that these metabolites correspond to the above-mentioned synthetic beta-oxidation products. This was further confirmed by the coelution of the corresponding methyl esters. Oxidative ozonolysis of the metabolically produced 16-carboxy-17,18,19,20-tetranor-14,15-dihydro-N-acetyl LTE4 (major metabolite) confirmed the absence of the 14,15-unsaturation. The presence of these metabolites indicates that peptide leukotrienes undergo N-acetylation followed by omega and subsequent beta-oxidation in the anesthetized rat.
Lipoxin synthesis by arachidonate 12-lipoxygenase purified from porcine leukocytes.
Arachidonate 12-lipoxygenase purified from porcine leukocytes shows 14R-oxygenase and 14, 15-leukotriene A synthase activities with 15-hydroperoxy-arachidonic acid as substrate. The enzyme transformed 5, 15-dihydroperoxy-arachidonic acid to several compounds with a conjugated tetraene. A major product was identified as 5S, 14R, 15S-trihydroperoxy-6, 10, 12-trans-8-cis-eicosatetraenoic acid, which was reduced to 5S, 14R, 15S-8-cis-lipoxin B. A requirement of molecular oxygen and the results of H2(18)O experiments suggested that formation of the latter compound was attributed mostly to the 14R-oxygenase activity of the enzyme. There were several other minor products identified as lipoxin A and B isomers. They were produced presumably by hydrolysis of 14, 15-epoxy compound formed by the leukotriene A synthase activity of 12-lipoxygenase.
Formation of lipoxin B by the pure reticulocyte lipoxygenase via sequential oxygenation of the substrate.
The pure reticulocyte lipoxygenase converts 15LS-hydroxy-5,8,11,13(Z,Z,Z,E)-icosatetraenoic acid (15LS-HETE) methyl ester to a complex mixture of products containing 5DS,14LR,15LS-trihydro(pero)xy-6E,++ +8Z,10E,12E-icosatetraenoate methyl ester (lipoxin B methyl ester), 5DS,15LS-DiH(P)ETE methyl ester and four 8,15LS-DiH(P)ETE methyl ester isomers [DiH(P)ETE = dihydro(pero)xy-icosatetraenoic acid]. After a short incubation period (15 min) 5DS,15LS-DiH(P)ETE methyl ester was found to be the main product, whereas after a 3-h incubation lipoxin B methyl ester was the predominant product. The reaction shows a remarkable stereoselectivity since only small amounts of other trihydroxy tetraenes are formed. Anaerobiosis, heat inactivation of the enzyme, or incubation in the presence of lipoxygenase inhibitors (icosatetraynoic acid, nordihydroguaiaretic acid) completely abolished the reaction. The complete steric structure of the major tetraene product (lipoxin B methyl ester) was established by ultraviolet spectroscopy, HPLC on four different types of columns, gas chromatography/mass spectrometry, gas/liquid chromatography of the ozonolysis fragments of the menthoxycarbonyl derivatives, and by 400-MHz 1H-NMR. Atmospheric oxygen was incorporated at carbon-5 and carbon-14 into the major product. 5DS,15LS-DiH(P)ETE methyl ester was shown to be an intermediate in the synthesis. Lipoxin B was also formed during the oxygenation of arachidonic acid, 15LS-HETE and 5DS,15LS-DiHETE. The results presented here indicate that lipoxin B can be formed by pure lipoxygenases via a sequential oxygenation of arachidonic acid or its hydro(pero)xy derivatives.
Evidence of in-vivo omega-oxidation of peptide leukotrienes in the rat: biliary excretion of 20-CO2H N-acetyl LTE4.
In a previous study in our laboratory it was observed that after [3H] LTC4 administration (luCi/kg i.v.) to the anesthetized rat, significant amounts of injected radioactivity (approximately 25%) were associated with previously unidentified biliary polar metabolite(s). In the present study we describe the isolation and characterization of the predominant polar metabolite. Rats were injected with synthetic LTC4 (20 microgram/kg i.v.) and bile collected over 30 min. After extraction and purification (2 step RP-HPLC procedure), the retention time of the metabolite was compared (plus coinjections) and found to be identical with synthetic 20-CO2H N-Ac LTE4 in two RP-HPLC systems. Also, the UV spectrum of the biologically derived metabolite was compared and found identical to the synthetic material, giving a characteristic conjugated triene absorption in the UV with a max of 281 nm and shoulders at 270 and 290 nm. Further, the trimethyl ester derivative of the metabolite showed identical chromatographic behaviors in 2 reverse and 2 normal phase HPLC systems compared with synthetic 20-CO2H N-Ac LTE4 trimethyl ester. We conclude omega-oxidation of peptide leukotrienes occurs in the rat and that 20-CO2H N-Ac LTE4 is an in vivo product of LTC4 metabolism.
Metabolism and excretion of peptide leukotrienes in the anesthetized rat.
The metabolism and excretion of the peptide leukotrienes C4, D4, E4 and N-acetylleukotriene E4 have been studied in the anesthetized rat. The intravenous administration of [3H]leukotriene C4 (2.6 X 10(-11) mol/kg) showed a rapid clearance of radioactivity from the blood and a time-related biliary excretion, recovering 69 +/- 1.6% (n = 6) over 60 min. Less than 1% of total radioactivity was recovered in the urine over the same time period. Similarly, the intravenous administration of [3H]leukotriene D4 (2.5 X 10(-11) mol/kg), [3H]leukotriene E4 (2.5 X 10(-11) mol/kg) and N-acetyl[3H]leukotriene E4 (2.1 X 10(-11) mol/kg) showed a 62 +/- 7.5% (n = 4), 52 +/- 1.5% (n = 4) and 37 +/- 4.6% (n = 5) biliary recovery of radioactivity, respectively, after 60 min. Examination of bile identified leukotriene D4 and N-acetylleukotriene E4 as the main products, although substantial radioactivity, which probably represents unidentified polar products, was present at the solvent fronts of the reverse-phase HPLC. Time course studies indicated a relatively rapid conversion of leukotriene C4 to leukotriene D4, while leukotriene D4 metabolism appeared to be much slower. Leukotriene E4 was a minor product, suggesting that the N-acetylation process is rapid. Incubation of [3H]leukotriene C4 in rat plasma and whole blood in vitro resulted in a slow conversion of leukotriene C4 to leukotriene D4 and leukotriene E4 only. These data suggest that the majority of the leukotriene metabolism and excretion in vivo in the anesthetized rat occurs predominantly in the hepatic system. We conclude that this model is suitable for the measurement of in vivo production of peptide leukotrienes.
Stereochemical requirements for substrate specificity of LTB4 20-hydroxylase.
LTB4 20-hydroxylase (P-450LTB) is the cytochrome P-450 in the microsomes of human polymorphonuclear leukocytes that catalyzes the omega-oxidation of leukotriene B4 (LTB4) to 20-OH LTB4. The activity of P-450LTB for LTB4 compared to isomers and analogs of LTB4 at a concentration of 0.3 microM revealed a preference of P-450LTB for both the triene bond configuration of LTB4 and for the chirality of the 5S and 12R hydroxyl groups. 15S-Hydroxyeicosatetraenoic acid, 8(R/S), 15S-dihydroxy-5-cis-9,11,13-trans-eicosatetraenoic acid, 8R,15S-dihydroxy-5,13-cis-9,11-trans-eicosatetraenoic acid, and 5S,15S-dihydroxy-6,13-trans-8,11-cis-eicosatetraenoic acid were each not subject to omega-oxidation, indicating a negative effect of the presence of a 15-hydroxyl group on substrate recognition. At a concentration of 1.5 microM, 12R- and 12S-hydroxyeicosatetraenoic acid were converted to their respective 20-OH derivatives at rates that were 34.2 +/- 11.6% (mean +/- S.D., n = 3) and 3.5 +/- 4.3% (mean +/- S.D., n = 4), respectively, of that of LTB4 to 20-OH LTB4, further indicating that P-450LTB can distinguish the chirality of the 12-hydroxyl group. The lower Km of LTB4 (2.0 microM), as compared to those of its 6-trans-12-epi isomer (3.8 microM) and 5-epi-LTB4 (6.6 microM) confirmed the preference of P-450LTB for the specific triene bond structure of LTB4 and its preference for the chirality of the hydroxyl groups of LTB4 within this structurally related class of molecules. At equal 1.5-microM concentrations, LTB4 completely inhibited the omega-oxidation of all other substrates and partially suppressed that of leukotriene B5, consistent with the lower Km of LTB4 and indicating that P-450LTB catalyzed the omega-oxidation of all substrates. Thus, P-450LTB is a novel cytochrome P-450 of human polymorphonuclear leukocytes with substrate recognition determined by the triene bond configuration and the chirality of the hydroxyl groups.
Lipoxin synthesis by arachidonate 5-lipoxygenase purified from porcine leukocytes.
Arachidonate 5-lipoxygenase purified from porcine leukocytes produced several more polar compounds from 5,15-dihydroperoxy-eicosatetraenoic acid added as such or generated from 15-hydroperoxy acid. These polar products with absorption maxima at 301-302 nm and shoulders at 289 nm and 316-317 nm were identified as 5S,6R,15S-11-cis-lipoxin A and its 6-epimer, all-trans-lipoxin A isomers, and all-trans-lipoxin B isomers. Most of these lipoxins were presumably degradation products of a 5,6-epoxy intermediate formed by the catalysis of leukotriene A synthase, an integral part of 5-lipoxygenase. The rate of the enzymatic lipoxin synthesis from 15-hydroperoxy acid was about 6% of arachidonate 5-oxygenation.
Analogs of leukotriene B4: effects of modification of the hydroxyl groups on leukocyte aggregation and binding to leukocyte leukotriene B4 receptors.
The syntheses and agonist and binding activities of 5(S)-hydroxy- 6(Z), 8(E), 10(E), 14(Z)-eicosatetraenoic acid (12-deoxy LTB4), 5(S), 12(S)-dihydroxy-6(Z), 8(E), 10(E), 14(Z)-eicosatetraenoic acid (12-epi LTB4), 12(R)-hydroxy-6(Z), 8(E), 10(E), 14(Z)-eicosatetraenoic acid (5-deoxy LTB4), 5(R), 12(S)-dihydroxy-6(Z), 8(E), 10(E), 14(Z)-eicosatetraenoic acid (5-epi LTB4), 6(Z), 8(E), 10(E), 14(Z)-eicosatetraenoic acid (5, 12-deoxy LTB4) are described. These leukotriene B4 analogs were all able to aggregate rat leukocytes and compete with [3H]-leukotriene B4 for binding to rat and human leukocyte leukotriene B4 receptors with varying efficacy. The analog in which the 12-hydroxyl group was removed was severely reduced both in agonist action (aggregation) and binding. The epimeric 12-hydroxyl analog demonstrated better agonist and binding properties than the analog without a hydroxyl at this position. In contrast, in the case of the 5-hydroxyl the epimeric hydroxyl analog had greatly reduced agonist and binding activities while the 5-deoxy analog demonstrated potency only several fold less than leukotriene B4 itself. The dideoxy leukotriene B4 analog was more than a thousand fold less active than leukotriene B4 as an agonist and in binding to the leukotriene B4 receptor. These results show that binding to the leukocyte leukotriene B4 receptor requires a hydroxyl group at the 12 position in either stereochemical orientation but that the presence of a hydroxyl at the 5 position is less important. However, the epimeric C5 leukotriene B4 analog clearly interacts unfavourably with the binding site of the leukotriene B4 receptor.
Biliary and urinary excretion of peptide leukotrienes in the domestic pig.
The metabolism of leukotriene (LT)C4 and its major routes of elimination in vivo have been studied in four anesthetized domestic pigs administered intravenous [3H]-LTC4 (0.5 microCi/kg). The kinetic profile of LTC4 in the blood was followed for 60 min after administration while the biliary and urinary excretion of LTC4 and its metabolites were determined over a 120 min interval. The total recovery of radioactivity in bile and urine was 45% +/- 1 (n = 3) and 18% (n = 2) respectively. Examination of the radioactive metabolites in bile showed LTD4 (44% of biliary content) and LTE4 (21% of biliary content) as the major identified lipoxygenase products at t 1/2 (27 min). The only identified cysteinyl leukotriene observed in the urine was LTE4 (13% of urinary content). In both bile and urine substantial amounts of radioactivity were detected at the solvent front of the reverse phase chromatographic system indicating the presence of additional unidentified metabolites. We suggest that measurement of metabolites using these sampling methods may be useful for the detection and measurement of peptide leukotriene production in vivo.
Dioxygenase and leukotriene A synthase activities of arachidonate 5- and 12-lipoxygenases purified from porcine leukocytes.
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Recent progress in the chemistry and biochemistry of lipoxygenase products: the lipoxins.
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The lipoxins: determination of their biosynthesis.
The stereochemistry and double bond geometry of a novel series of leukocyte derived arachidonic acid metabolites, the lipoxins, was determined by comparison to pure unambiguous synthetic standards. The lipoxins were found to be a mixture of four lipoxin A isomers and two lipoxin B isomers. In determining the biosynthesis of these compounds, they were shown to be formed via a tetraene epoxide. In addition, it was shown that all of the lipoxin isomers formed by the incubation of 15-HPETE with human leukocytes were also formed by non enzymatic hydrolysis of this tetraene epoxide.
Trihydroxytetraene metabolites of arachidonic acid: the lipoxins.
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The lipoxins. Stereochemical identification and determination of their biosynthesis.
The stereochemistry and double bond geometry of a novel series of leukocyte-derived arachidonic acid metabolites, the lipoxins, was determined by comparison to pure unambiguous synthetic standards. The lipoxins were found to be a mixture of four lipoxin A isomers and two lipoxin B isomers. In determining the biosynthesis of these compounds, they were shown to be formed via a tetraene epoxide. In addition, it was shown that all of the lipoxin isomers formed by the incubation of 15-hydroperoxyeicosatetraenoic acid with human leukocytes were also formed by nonenzymatic hydrolysis of this tetraene epoxide.
Catabolism of leukotriene A4 into B4, C4, and D4 by rat liver subcellular fractions.
[3H]Leukotriene A4 was incubated with various subcellular fractions of rat liver homogenates. After solvent extraction and purification on C18 Sep-Pak cartridges, tritiated products migrating on reversed-phase HPLC with authentic unlabelled leukotriene C4, D4 and B4 were observed. The identity of leukotriene C4 was confirmed through enzymatic conversion into D4 by gamma-glutamyl transpeptidase as well as by bioassay on the rat stomach fundus after HPLC purification. The contractile response to the extracted material was blocked by the SRS antagonist, FPL 55712. Leukotriene B4 synthesis was located in the 100 000 X g supernatant, while C4 synthesis was present in the corresponding pellet. Leukotriene C4 formation was enhanced when reduced glutathione was supplemented in the incubation medium. These results demonstrate the presence in rat liver of various enzymatic steps in leukotriene A4 catabolism.
Actions of synthetic leukotrienes on platelets and blood vessels in the anesthetised pig: the release of a platelet derived vasodilator.
The actions of leukotrienes (LT's) C4, D4, E4 and F4 have been investigated in the perfused hind-limb of the anesthetized pig. In the blood perfused hind limb LTC4, D4 and E4 increased the perfusion pressure in a dose-dependent fashion whereas LTF4 decreased perfusion pressure. In the Tyrode perfused hind limb all LT's increased perfusion pressure (rank order potency LTC4 = LTD4 much greater than LTF4). The actions of LTF4 were not affected by a wide variety of pharmacological treatments, including indomethacin, methysergide and FPL-55712. The LT's aggregated porcine platelets (rank order potency LTC4 greater than LTF4 greater than LTD4) and induced the release of a platelet-derived vasodilatory mediator. The results provide pharmacological evidence of specific leukotriene receptors in vivo and that leukotrienes can independently modulate blood flow. These data suggest that important interactions may occur between platelets, the arachidonate lipoxygenase products and platelet-derived substances in response to inflammatory stimuli in the cardiovascular system.
Chemical and enzymatic syntheses of lipoxin A: stereochemical assignment of natural lipoxin A and its possible biosynthesis.
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