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Isoleukotrienes Are Biologically Active Free Radical Products of Lipid Peroxidation

1995; Elsevier BV; Volume: 270; Issue: 29 Linguagem: Inglês

10.1074/jbc.270.29.17273

1083-351X

Kathleen A. Harrison, Robert C. Murphy,

Vitamin C and Antioxidants Research

The free radical oxidation of arachidonic acid esterified to glycerophospholipids is known to generate complex metabolites, termed isoprostanes, that share structural features of prostaglandins derived from prostaglandin H2 synthase. Furthermore, certain isoprostanes have been found to exert biological activity through endogenous receptors on cell surfaces. Using mass spectrometry and ancillary techniques, the free radical oxidation of 1-hexadecanoyl-2-arachidonoyl-glycerophosphocholine was studied in the search for products of arachidonic acid isomeric to the leukotrienes that are derived from 5-lipoxygenase-catalyzed metabolism of arachidonic acid. Several conjugated triene metabolites were chromatographically separated from known 5-lipoxygenase products and structures characterized as 5,12-dihydroxy-6,8,10,14-eicosatetraenoic acid esterified to the glycerophosphocholine backbone. We have termed these products as B4-isoleukotrienes. Following saponification some, but not all, B4-isoleukotrienes were found to exert biological activity in elevating intracellular calcium in Indo-1-loaded human polymorphonuclear leukocytes. This activity could be blocked by a leukotriene B4 receptor antagonist. An EC50 of approximately 30 nM was determined for one unique B4-isoleukotriene with a relative retention index of 2.54. We have shown that free radical processes can lead to the formation of biologically active isoleukotrienes in glycerophosphocholine liposomes, and we propose that B4-isoleukotrienes may also be formed in membrane glycerophospholipids as a result of lipid peroxidation during tissue injury. Such B4-isoleukotrienes could then mediate events of tissue damage through activation of leukotriene B4 receptors on target cells. The free radical oxidation of arachidonic acid esterified to glycerophospholipids is known to generate complex metabolites, termed isoprostanes, that share structural features of prostaglandins derived from prostaglandin H2 synthase. Furthermore, certain isoprostanes have been found to exert biological activity through endogenous receptors on cell surfaces. Using mass spectrometry and ancillary techniques, the free radical oxidation of 1-hexadecanoyl-2-arachidonoyl-glycerophosphocholine was studied in the search for products of arachidonic acid isomeric to the leukotrienes that are derived from 5-lipoxygenase-catalyzed metabolism of arachidonic acid. Several conjugated triene metabolites were chromatographically separated from known 5-lipoxygenase products and structures characterized as 5,12-dihydroxy-6,8,10,14-eicosatetraenoic acid esterified to the glycerophosphocholine backbone. We have termed these products as B4-isoleukotrienes. Following saponification some, but not all, B4-isoleukotrienes were found to exert biological activity in elevating intracellular calcium in Indo-1-loaded human polymorphonuclear leukocytes. This activity could be blocked by a leukotriene B4 receptor antagonist. An EC50 of approximately 30 nM was determined for one unique B4-isoleukotriene with a relative retention index of 2.54. We have shown that free radical processes can lead to the formation of biologically active isoleukotrienes in glycerophosphocholine liposomes, and we propose that B4-isoleukotrienes may also be formed in membrane glycerophospholipids as a result of lipid peroxidation during tissue injury. Such B4-isoleukotrienes could then mediate events of tissue damage through activation of leukotriene B4 receptors on target cells. Oxygen free radicals are generated in vivo by a variety of enzymatic and nonenzymatic reactions(1Pryor W.A. Annu. Rev. Physiol. 1986; 48: 657-667Crossref PubMed Scopus (803) Google Scholar, 2Britton R.S. Bacon B.R. Hepatogastroenterology. 1994; 41: 343-348PubMed Google Scholar, 3Halliwell B. Am. J. Med. 1991; 91: 14S-22SAbstract Full Text PDF PubMed Scopus (850) Google Scholar). These reactive oxygen species are thought to play an important role in tissue damage characteristic of many diseases including atherosclerosis, inflammatory diseases, cancer, aging, and ischemia reperfusion injury(4Jaeschke H. Chem. Biol. Interact. 1991; 79: 115-136Crossref PubMed Scopus (303) Google Scholar, 5Halliwell B. Gutteridge J.M.C. Free Radicals in Biology and Medicine. Oxford University Press, Oxford, United Kingdom1989: 416-508Google Scholar, 6Tarr M. Samson F. Oxygen Free Radical in Tissue Injury. Birkhauser, Boston1993Crossref Google Scholar). While the exact biochemical mechanisms relating free radical generation to the pathology are unclear, most points of view consider profound alterations in tissue biochemistry as a result of lipid peroxidation, DNA damage, or irreversible alteration of proteins(7Floyd R.A. Carney J.M. Ann. Neural. 1992; 32: S22-S27Crossref PubMed Scopus (502) Google Scholar, 8Stadtman E.R. Free Radical Biol. Med. 1990; 9: 315-325Crossref PubMed Scopus (1035) Google Scholar, 9Halliwell B. Gutteridge J.M.C. Free Radicals in Biology and Medicine. Oxford University Press, Oxford1989: 188-276Google Scholar, 10Esterbauer H. Schaur R.J. Zollner H. Free Radicals Biol. Med. 1991; 11: 81-128Crossref PubMed Scopus (5960) Google Scholar, 11Kneepkens C.M. Lepage G. Roy C.C. Free Radicals Biol. Med. 1994; 17: 127-160Crossref PubMed Scopus (326) Google Scholar). Although lipid peroxidation leads to a large number of products, some of which are stable species derived from polyunsaturated fatty acyl substituents of phospholipids in membrane bilayers, the measurement of low molecular weight products such as pentane, malonyldialdehyde, and 4-hydroxynonenal have been the most widely measured oxidation products for studies of lipid peroxidation(12Shimizu T. Kondo K. Hayaishi O. Arch. Biochem. Biophys. 1981; 206: 271Crossref PubMed Scopus (105) Google Scholar, 13Basu A. Marnett L. Carcinogenesis. 1984; 4: 331Crossref Scopus (267) Google Scholar). It is likely, however, that these low molecular weight products result from extensive rearrangement of the initial oxidized phospholipid species and, as such, may not reflect initial oxidation events taking place at the lipid bilayer membrane level. Recently compounds that are isomeric to prostaglandins (isoprostanes) were discovered to be generated by free radical-mediated processes and whose formation was not catalyzed by the enzyme prostaglandin H synthase(14Morrow J.D. Hill K.E. Burk R.F. Nammour T.M. Badr K.F. Roberts L.J. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 9383-9387Crossref PubMed Scopus (1727) Google Scholar, 15Morrow J.D. Minton T.A. Mukundan C.R. Campbell M.D. Zackert W.E. Daniel V.C. Badr K.F. Blair I.A. Roberts II, L.J. J. Biol. Chem. 1994; 269: 4317-4326Abstract Full Text PDF PubMed Google Scholar). F2-isoprostanes (isomeric of prostaglandin F2α) were readily identified esterified to phospholipids in the liver of rats that had been treated with carbon tetrachloride in a model of free radical hepatotoxicity(16Kayganich-Harrison K.A. Rose D.M. Murphy R.C. Morrow J.D. Roberts L.J. J. Lipid Res. 1993; 34: 1229-1235Abstract Full Text PDF PubMed Google Scholar). One such free radical product, 8-epi prostaglandin F2α was also found to have significant biological activity as the free acid. This isoprostane was found to cause a potent constriction of the renal artery of the rat and reduced glomerular filtration rate in the rat kidney(17Morrow J.D. Minton T.A. Roberts II, L.J. Prostaglandins. 1992; 44: 155-163Crossref PubMed Scopus (219) Google Scholar). It was also found to have activity in other organs including the lung, where it constricted the pulmonary artery, but was a more potent constrictor of the bronchial airway in the isolated perfused lung(18Kang K.H. Morrow J.D. Roberts L.J. Newman J.H. Banerjee M. J. Appl. Physiol. 1993; 74: 460-465Crossref PubMed Google Scholar). Interestingly, these biological activities could be prevented or even reversed by a thromboxane A2 receptor antagonist(17Morrow J.D. Minton T.A. Roberts II, L.J. Prostaglandins. 1992; 44: 155-163Crossref PubMed Scopus (219) Google Scholar), suggesting that this F2-isoprostane was exerting its pharmacological effect through the endogenous receptor for thromboxane. These findings have led to the suggestion that isoprostanes may serve as lipid mediators of free radical-induced damage at the tissue level(15Morrow J.D. Minton T.A. Mukundan C.R. Campbell M.D. Zackert W.E. Daniel V.C. Badr K.F. Blair I.A. Roberts II, L.J. J. Biol. Chem. 1994; 269: 4317-4326Abstract Full Text PDF PubMed Google Scholar, 17Morrow J.D. Minton T.A. Roberts II, L.J. Prostaglandins. 1992; 44: 155-163Crossref PubMed Scopus (219) Google Scholar). Since the nonenzymatic, free radical-induced formation of F2-isoprostanes in vivo result from a free radical attack on arachidonoyl-containing phospholipids in vivo, it became of interest to investigate whether or not other complex molecules esterified to phospholipids could be formed by free radical reactions, in particular complex molecules structurally related to the leukotrienes. Leukotrienes are normally thought to be derived only from the 5-lipoxygenase pathway of arachidonic acid metabolism. To clarify whether or not such compounds could be formed, we investigated the free radical oxidation 1-hexadecanoyl-2-arachidonoyl-glycerophosphocholine liposomes and report that hydroxyl radical generated by a modified Fenton reaction (19Halliwell B. Gutteridge J.M. Biochem. J. 1984; 219: 1-14Crossref PubMed Scopus (4593) Google Scholar) led to the formation of numerous products including several isomers of leukotriene B4 (isoleukotrienes) that exhibited biological activity via the leukotriene B4 receptor. Leukotriene B4 and other eicosanoid standards were purchased from Cayman Chemical Co. (Ann Arbor, MI) and used without further purification. 1-Hexadecanoyl-2-arachidonoyl glycerophosphocholine was purchased from Avanti Polar Lipids (Alabaster, AL). Radiolabeled 1-hexadecanoyl-2[14C1]arachidonoyl glycerophosphocholine (57 mCi/mmol) was purchased from DuPont NEN. The LTB4 receptor antagonist LY223982 was a kind gift from Eli Lilly. All solvents used were of the highest available purity. Hydrogen peroxide (30%, w/v), copper(II) chloride, digitonin, ammonium acetate, EGTA, Trizma (Tris base), and phosphate-buffered saline tablets were purchased from Sigma or Aldrich. Sepralyte octadecylsilyl solid phase extraction packing material (40 μm) was purchased from Analytichem International (Harbor City, CA). Pentafluorobenzyl bromide, diisopropylethylamine, and 5% rhodium adsorbed on alumina powder were purchased from Aldrich. Bis-trimethylsilyl trifluoroacetamide was purchased from Supelco (Bellefonte, PA). Indo-1 acetoxymethyl ester was obtained from Calbiochem. Ten micromoles of 1-hexadecanoyl-2-arachidonoyl-GPCho 1The abbreviations used are: GPChoglycerophosphocholinePBSphosphate-buffered salineHPLChigh performance liquid chromatographyESIelectrospray ionizationMSmass spectrometryLTB4leukotriene B4GCgas chromatographyRRIrelative retention index. 1 in CHCl3 solution (10 mg/ml) was placed in an 8-ml screw cap glass tube. 1-Hexadecanoyl-2[14C1]arachidonoyl-GPCho (5 μCi) was also added to some reactions. The solvent was evaporated at room temperature under a stream of N2 gas. The phospholipid was immediately resuspended in 4.8 ml of 50 mM PBS, pH 7.3, by vortexing and then sonicating for 5 s at maximum power. H2O2 (30%, w/v), and 6 mM CuCl2 were added to the solution resulting in final concentrations of 600 mM and 100 μM, respectively. The solution was capped and heated to 37°C on a gently shaking water bath for 3 h. For some experiments reactions were allowed to continue at 5°C overnight before extraction. Progress of oxidation was monitored by UV absorbance at 235 nm for conjugated dienes and 270 nm for conjugated trienes in a 0.1% aliquot. glycerophosphocholine phosphate-buffered saline high performance liquid chromatography electrospray ionization mass spectrometry leukotriene B4 gas chromatography relative retention index. The reaction mixture was loaded onto 2 g of reverse phase 40-μm silica particles packed in a low pressure glass column. The column was preconditioned with 30 ml of methanol followed by 30 ml of PBS under slight pressure. Salts and remaining H2O2 were eluted with 30 ml of water. Oxidized GPCho was eluted with 15 ml of methanol into a 50-ml pear-shaped flask. The remaining 1-hexadecanoyl-2-arachidonoyl-GPCho was eluted with an additional 10 ml of methanol. Hydroperoxides or endoperoxides formed during the oxidation reaction were reduced with stannous chloride to prevent further rearrangement and degradation of these products. Stannous chloride (100 mM) was added to the methanol fraction containing the oxidized GPCho to a final concentration of 1 mM. The flask was placed on a rotary evaporator and the solvent removed to near dryness. The remaining liquid in the flask was rinsed into a test tube (∼1 ml) and injected directly onto the reverse-phase HPLC system. A Beckman (Berkeley, CA) ODS 5-μm 4.6 mm × 25-cm column with a Waters (Marlborough, MA) ODS Guard-pak precolumn was used to separate the oxidized GPCho. The solvent system (system A) consisted of 85% methanol, 1 mM ammonium acetate at 1.5 ml/min for 25 min, followed by a linear gradient to 100% methanol, 1 mM ammonium acetate over a 50-min period. Either a photodiode array detector (Hewlett-Packard 1090A) or a linear 206 scanning UV detector (Linear Instruments, Reno, NV) were used to continuously record UV spectra, scanning from 205 nm to 320 nm with a 1-nm step size. A fraction collector was used to collect effluent at 1-min intervals. For those experiments in which radiolabeled 1-hexadecanoyl-2-arachidonoyl GPCho was used, 10% of the oxidized GPCho products were separated on HPLC with a Flo-One Beta (Radiomatic, Riviera Beach, FL) radiochromatography detector connected to the effluent stream after the UV detector. Reverse phase HPLC fractions containing the conjugated triene chromophore were analyzed using electrospray ionization (ESI) mass spectrometry and tandem mass spectrometry. Flow injection was used to introduce 2 μl of the HPLC fractions at a flow rate of 10 μl/min with 85% methanol, 1 mM ammonium acetate as the mobile phase. The Sciex API III+ (Perkin-Elmer Sciex, Toronto, Canada) was operated in negative ion mode with an orifice voltage of −105 V in order to collisionally decompose the GPCho acetate adducts to [M − 15]− ions. Product ion spectra were obtained using a collision energy of 30 eV and collision gas thickness (argon) of 220 × 1012 molecules/cm2. Negative ion ESI mass spectra of the oxidized fatty acids obtained from hydrolysis of the oxidized GPCho were obtained using an orifice voltage of −60 V. For all ESI analyses, the curtain gas flow was 1.2 liter/min, the nebulizer pressure was 40 p.s.i., the turbospray flow was 7 liters/min, and the turbospray temperature was 400°C. Fractions from the first HPLC separation (system A) identified as containing components with a conjugated triene chromophore were saponified by addition of 0.5 ml of 1 N sodium hydroxide at room temperature for 1 h. The fractions were then acidified with 50 μl of 88% formic acid, and the methanol evaporated under vacuum. The fractions were reconstituted in 1 ml of 30% methanol, 70% water (0.05% acetic acid), pH 5.7, for injection into the HPLC system. Spectra of peaks eluting from the Beckman ODS 5-μm 4.6 mm × 25-cm column with a Waters ODS Guard-pak precolumn were continuously collected using a photodiode array detector. The HPLC was operated with a three-step gradient (system B) starting at 40% B and ramping to 55% B in 6 min, then to 61% B in 15 min, and to 100% B in 5 min, where B = methanol:acetonitrile (35:65) and A = 0.05% acetic acid, adjusted to pH 5.7 with ammonium hydroxide. The flow rate was 1 ml/min, and fractions were automatically collected at 0.5 min/tube. The elution of each component relative to prostaglandin B2 and LTB4 external standards was calculated by the following equation for relative retention index (RRI). RRIretention time unknown - retention time PGB2retention time LTB4 - retention time PGB2+1(Eq. 1) HPLC analysis of the external standards was run immediately after the analysis of oxidized eicosanoids. Changes in neutrophil intracellular calcium levels were determined by measuring the fluorescence of Indo-1 as described previously (20Powell W.S. Gravel S. MacLeod R.J. Mills E. Hashefi M. J. Biol. Chem. 1993; 268: 9280-9286Abstract Full Text PDF PubMed Google Scholar) with minor changes. The intracellular calcium levels were calculated as described (21Grynkiewicz G. Poenie M. Tsien R.Y. J. Biol. Chem. 1985; 260: 3440-3450Abstract Full Text PDF PubMed Scopus (80) Google Scholar) using a dissociation constant of 250 nM for Indo-1/Ca2+ complex. In some cases, the cells were preincubated with 10 μM LTB4 receptor antagonist, LY223982, prior to the addition of agonists. The remainder of the HPLC system B fractions containing the hydrolyzed fatty acids were analyzed both by electron capture negative ion mass spectrometry and by electron impact ionization mass spectrometry using a Finnigan SSQ mass spectrometer (Finnigan Corp., San Jose, CA) interfaced with a capillary gas chromatograph column. For electron capture GC/MS, a portion of the fractions were derivatized as pentafluorobenzyl esters and trimethylsilyl ethers as described previously(22Wheelan P. Zirrolli J.A. Murphy R.C. J. Am. Soc. Mass Spectrom. 1995; 6: 40-51Crossref PubMed Scopus (34) Google Scholar). Prior to electron ionization GC/MS, samples were hydrogenated by bubbling hydrogen gas through a methanol solution of the sample for 30 min using rhodium adsorbed on alumina as the catalyst (∼1 mg). The samples were then derivatized as pentafluorobenzyl esters trimethylsilyl ethers. Reverse phase HPLC separation of the oxidized 1-hexadecanoyl-2-arachidonoyl-GPCho revealed the presence of a large number of products. The major component in the mixture remained unreacted 1-hexadecanoyl-2-arachidonoyl-glycerophosphocholine, as indicated by the absorbance profile at 205 nm and elution at 78 min (Fig. 1A). The elution of conjugated trienes was indicated by absorbance at 270 nm (Fig. 1B), and many components absorbing at this wavelength were detected having significantly less lipophilicity than the starting material. The UV spectra of several components eluting between 44 and 54 min were suggestive of the characteristic vibronic UV absorption of conjugated trienes, as illustrated by the component eluting at 53 min in this HPLC separation (Fig. 1B, inset). The UV absorption profile at 235 nm (data not shown) revealed elution of several conjugated diene oxidized products in this same general area of the chromatogram. Aliquots of each fraction collected during the HPLC separation were analyzed by electrospray ionization (ESI) mass spectrometry, which yields abundant [M − 15]− ions for the lipid glycerophosphocholine molecular species(23Kerwin J.L. Tuininga A.R. Ericsson L.H. J. Lipid Res. 1994; 35: 1102-1114Abstract Full Text PDF PubMed Google Scholar). For example, fraction 53 yielded abundant [M − 15]− ions at m/z 798.6 and 814.7. These [M − 15]− ions were then selected for subsequent tandem mass spectrometry. Collision-induced decomposition of the ion at m/z 798.6 from fraction 53 yielded two carboxylate anions at m/z 255 (characteristic for hexadecanoate) and m/z 335 (corresponding to the addition of two oxygen atoms to the arachidonate carboxylate anion) consistent with the carboxylate anion for a dihydroxyeicosatetraenoic acid (Fig. 1C). The ion at m/z 480 corresponded to the loss of the sn-2 substituent as a neutral ketene, confirming that the dioxygenated arachidonoyl moiety was esterified at sn-2. The ions at m/z 317 and 195 likely resulted from secondary fragmentation of m/z 335. Tandem mass spectrometric analysis of HPLC fractions between 44 and 54 min revealed elution of several dioxygenated eicosatetraenoic acids esterified at the sn-2 position of phosphatidylcholine. The oxidized GPChos in the fractions between 44 and 54 min (Fig. 1A) were individually saponified and the liberated oxidized fatty acids were purified by the second reverse phase HPLC, system B. Components were observed in several fractions that had UV spectra with maximum absorption at 270 nm and vibronic bands 10 nm on either side at 260 and 280 nm, characteristic of a conjugated triene (Table 1). Each HPLC fraction (system B) was tested for its ability to elicit an increase in intracellular calcium from human polymorphonuclear leukocytes loaded with the fluorescent dye Indo-1 to screen for components with biological activity. HPLC separation of the free acids liberated from phospholipid fraction 53 (system A) is shown in Fig. 2A with the elution of a single component at 15.3 min (RRI = 2.54) absorbing at 270 nm (Fig. 2A, inset). The 0.5-ml fraction collected between 15 and 15.5 min was also found to have the highest level of activity in elevating intracellular calcium in the human neutrophil (Fig. 2B). Other closely eluting molecules maximizing in adjacent fractions were also present in this sample; however, these components did not have the characteristic UV chromophore of a conjugated triene (data not shown) but may have biological activity.TABLE I Open table in a new tab Negative ion ESI/MS of the HPLC fractions (system B) containing conjugated triene free acids consistently revealed [M − H]− ions at m/z 335, consistent with the carboxylate anion expected for dioxygenated arachidonic acid. Collision-induced decomposition of m/z 335 from these fractions yielded numerous product ions that were similar but often not identical to those observed following collision-induced decomposition of LTB4 (data not shown). Collision-induced decomposition of m/z 335 from the component eluting at 15.3 min (Fig. 2A) yielded the MS/MS spectrum shown in Fig. 3A with characteristic ions at m/z 59, 129, 195, and 317, all of which are also observed upon collision-induced decomposition of m/z 335 from LTB4, 6-trans-LTB4, and 6-trans-12-epi-LTB4. This collision-induced decomposition mass spectrum differed significantly from that of other dihydroxy eicosanoids such as 8,15-, 5,6-, or 5,15-dihydroxyeicosatetraenoic acid isomers (data not shown). This mass spectral data was consistent with a 20-carbon fatty acid containing four double bonds and two hydroxyl substituents. The covalent backbone of the molecule was established following catalytic reduction of this metabolite using hydrogen and Rh/Al2O3 followed by derivatization to the pentafluorobenzyl ester trimethylsilyl ether. The electron ionization mass spectrum (Fig. 3B) clearly revealed the presence of a saturated 20-carbon fatty acid derivative with two hydroxyl groups as trimethylsilyl ethers. The α-cleavage ions m/z 555 and 215 supported assignment of a 12-hydroxy substituent, and m/z 369 and 401 supported assignment of a 5-hydroxy substituent(24Murphy R.C. Handbook of Lipid Research: Mass Spectrometry of Lipids. Plenum Press, New York1993: 106-108Google Scholar). A conjugated triene could only exist between the hydroxyl substituents at carbon atoms 5 and 12, placing the triene at carbons 6, 8, and 10. While the position of an isolated double bond at carbon 14 was not unambiguously assigned, this was the original position of the ω-6 double bond in arachidonic acid and this portion of the molecule was likely not altered by the oxidation process. The structure of this free radical product was thus established as 5,12-dihydroxy-6,8,10,14-eicosatetraenoic acid, a B4-isoleukotriene. The oxidized phospholipid from which this dihydroxyeicosanoid was obtained was therefore 1-hexadecanoyl-2-[5,12-dihydroxy-6,8,10,14-eicosatetraenoyl]glycerophosphocholine (see Fig. SI).Figure SI:Scheme I.View Large Image Figure ViewerDownload Hi-res image Download (PPT) As shown in Table 1, several conjugated triene eicosanoids were products of the free radical oxidation of 1-hexadecanoyl-2-arachidonoyl-GPCho. Several of these eicosanoids were found to stimulate an increase in intracellular free calcium ion in the human neutrophil. The absolute quantity used in the neutrophil assay differed for each sample since an equal proportion (16%) of each HPLC fraction was tested for biological activity and the absolute yield of each eicosanoid free radical product varied. In order to compare activities in the absence of complete dose-response curves, the biological activity in Table 1 is expressed as the natural logarithm of the increase in intracellular calcium concentration (nM) per nanomolar concentration of eicosanoid tested. Some compounds displayed no activity in this assay. For example, the component with the HPLC relative retention index at 1.66 elicited no increase in intracellular free calcium ion when tested at a concentration of 28.2 nM (54 pmol). In contrast, the component that eluted with a relative retention index of 1.77 caused an increase of 626 nM [Ca2+]i when tested with 38 pmol. The response of Indo-1-labeled neutrophils to LTB4 (7 pmol) and the isolated isoleukotriene with an RRI = 1.86 (68 pmol) is shown in Fig. 4. Both compounds elicited a large increase in free intracellular calcium within the human neutrophil, although LTB4 was more than 10-fold more potent in this response. In contrast, the dual lipoxygenase product (5S,12S)-dihydroxyeicosatetraenoic acid in much larger quantity (90 pmol) elicited only a 43 nM increase in intracellular free calcium ion (data not shown). Further investigation of the pharmacologic effect of leukotrienes on the human neutrophil revealed that elevation of intracellular calcium ion could be blocked by administration of 10 μM LTB4 receptor antagonist LY223982(27Jackson W.T. Boyd R.J. Froelich L.L. Mallett B.E. Gapinski D.M. J. Pharmacol. Exp. Ther. 1992; 263: 1009-1014PubMed Google Scholar). As shown in Fig. 4, both responses from the component with relative retention index = 1.86 and LTB4 were completely attenuated by the LTB4 receptor antagonist LY223982. The LTB4 receptor antagonist also blocked the biological response elicited by eicosanoids with relative retention index of 2.54 and 1.77 (data not shown). There was sufficient quantity of the isoleukotriene eluting with a relative retention index of 2.54 (Fig. 2A) for a dose-response study (Fig. 5). An EC50 of 30 nM was calculated for this eicosanoid product. This is approximately 100-fold less than that observed for LTB4 itself, which has an EC50 of 0.3-1 nM(25Kobayashi Y. Shimazaki T. Kawajiri K. Shimizu T. Seyama Y. Sato F. Biochim. Biophys. Acta. 1994; 1215: 280-284Crossref PubMed Scopus (8) Google Scholar, 26Seya A. Terano Y. Tamura S. Yoshida S. Prostaglandins Leukotrienes Essent. Fatty Acids. 1988; 34: 37-43Abstract Full Text PDF PubMed Scopus (27) Google Scholar). Free radical oxidation of arachidonic acid esterified to glycerophospholipids is known to result in a host of oxidized intermediates. The recent discovery of the isoprostanes as free radical oxidation products of arachidonic acid and arachidonate-containing glycerophospholipids has emphasized that complex rearrangements of intermediate peroxy radical or hydroperoxides, formed as initial oxidation products, can take place. Described here is a new family of free radical-generated eicosanoids derived from arachidonoyl phospholipids. Several dihydroxy eicosanoids were observed, including several that contain a conjugated triene structural feature similar to that observed in the enzymatic products of 5-lipoxygenase. Detailed structural studies of an abundant, biologically active component (RRI = 2.54) revealed this eicosanoid to be a 5,12-dihydroxy-6,8,10,14-eicosatetraenoic acid. Therefore, we have termed this conjugated triene dihydroxy eicosanoids as a "B4-isoleukotriene" as a free radical product of lipid peroxidation that resembles enzymatically produced LTB4. Several free radical mechanisms are possible by which such dihydroxy-conjugated trienes could be formed from arachidonic acid esterified to phospholipids. It is most likely that two separate radical abstraction events removing the bisallylic hydrogen atoms on carbon atoms 7 and 10 occurred and the addition of molecular oxygen resulted at carbon atoms 5 and 12 of the arachidonic acid backbone. One interesting possibility is an intermediate formation of a leukotriene A4-like structure as a non-free radical intermediate present in the phospholipid. Hydrolysis of such a conjugated triene epoxide with water would yield isoleukotrienes as 5,12-dihydroxyeicosatetraenoic acids. Several B4-isoleukotrienes were found to be potent agonists stimulating the elevation of intracellular free calcium ion in the human neutrophil. Furthermore, this action could be blocked by the LTB4 receptor antagonist LY223982(27Jackson W.T. Boyd R.J. Froelich L.L. Mallett B.E. Gapinski D.M. J. Pharmacol. Exp. Ther. 1992; 263: 1009-1014PubMed Google Scholar). While the potency of the B4-isoleukotriene that was studied in greatest detail (RRI = 2.54) was found to be somewhat less than that for LTB4 itself, it is similar to that found for other chemotactic substances such as the peptide fMLP(28White J.R. Naccache P.H. Molski T.F. Borgeat P. Sha'afi R.I. Biochem. Biophys. Res. Commun. 1983; 113: 44-50Crossref PubMed Scopus (132) Google Scholar, 29Omann G.M. Traynor A.E. Harris A.L. Sklar L.A. J. Immunol. 1987; 138: 2626-2632PubMed Google Scholar, 30Lazzari K.G. Proto P.J. Simons E.R. J. Biol. Chem. 1986; 261: 9710-9713Abstract Full Text PDF PubMed Google Scholar). This B4-isoleukotriene and others described here, if formed in vivo, would have sufficient potency to play an important role in activating or priming neutrophils for a respiratory burst or priming neutrophils for leukotriene production by the 5-lipoxygenase pathway. 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A wide variation in activity was observed for such isomers, suggesting certain structural features of LTB4 are important for binding and biological activity, which include a cis configuration of the double bond at carbon-6 and stereochemistry of the 12-hydroxyl substituent. The geometry of the double bonds in the conjugated triene moiety is also quite important for receptor recognition and it is noteworthy that the trans, cis, trans configuration of the double bonds at carbons 6, 8, and 10 confers tighter binding than those isomers having the all trans configuration in the conjugated triene moiety(39Lee T.H. Mencia-Huerta J.-M. Shih C. Corey E.J. Lewis R.A. Austen K.F. J. Biol. Chem. 1984; 259: 2383-2389Abstract Full Text PDF PubMed Google Scholar). However, many of the synthetic isomers possess profound activity in many systems. The exact stereochemistry of the biologically active B4-isoleukotrienes is currently under investigation. B4-Isoleukotrienes are a family of free radical-generated eicosanoids derived from arachidonoyl glycerophospholipids. These free radical products of lipid peroxidation resemble enzymatically produced LTB4 in both structure and biological activity. As such they could serve as lipid mediators of cellular free radical damage in tissues exerting an effect by way of the LTB4 receptor.