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• W2074616170 abstract "In previous studies we observed that normal human epidermis forms 12-oxo-eicosatetraenoic acid (12-oxo-ETE) and hepoxilin B3 (HxB3) as major eicosanoids, both being elevated in psoriasis. We also observed that normal epidermis, in a reaction probably catalyzed by 12-lipoxygenase, only synthesize one of the two possible 10-hydroxy epimers of HxB3. We have now extended these previous studies investigating further transformation of HxB3 into trioxilin B3 (TrXB3) and esterification of both into phospholipids. Phospholipids were extracted from normal epidermis and from psoriatic scales. A combination of high performance liquid chromatography and gas chromatography-mass spectrometry analysis demonstrated the occurrence of HxB3 and TrXB3 in the phospholipids of psoriatic lesions. Alkaline- and phospholipase-A2-mediated hydrolysis of the phospholipids yielded similar quantities of both HxB3 and TrXB3 indicating their preference for the sn-2 position of glycerophospholipids. The thin layer chromatography analysis of the phospholipid classes after incubation of epidermal cells with [14C]-labeled HxB3, TrXB3, 12-hydroxy-eicosatetraenoic acid (12-HETE), 12-oxo-ETE, or 15-HETE showed that 12-HETE was the most esterified (12-HETE > 15-HETE > TrXB3 > 12-oxo-ETE > HxB3). HxB3 and TrXB3 were mainly esterified in phosphatidyl-choline and phosphatidyl-ethanolamine. HxB3 was also enzymatically converted into TrXB3 in vitro. HxB3 epoxide hydrolase-like activity was not observed when boiled tissue was incubated with [14C]-HxB3, this activity being located in the cytosol fraction (100,000 × g supernatant) of fresh tissue. These findings suggest that in vivo some part of HxB3 is transformed into TrXB3 and both compounds are partially incorporated into the phospholipids. In previous studies we observed that normal human epidermis forms 12-oxo-eicosatetraenoic acid (12-oxo-ETE) and hepoxilin B3 (HxB3) as major eicosanoids, both being elevated in psoriasis. We also observed that normal epidermis, in a reaction probably catalyzed by 12-lipoxygenase, only synthesize one of the two possible 10-hydroxy epimers of HxB3. We have now extended these previous studies investigating further transformation of HxB3 into trioxilin B3 (TrXB3) and esterification of both into phospholipids. Phospholipids were extracted from normal epidermis and from psoriatic scales. A combination of high performance liquid chromatography and gas chromatography-mass spectrometry analysis demonstrated the occurrence of HxB3 and TrXB3 in the phospholipids of psoriatic lesions. Alkaline- and phospholipase-A2-mediated hydrolysis of the phospholipids yielded similar quantities of both HxB3 and TrXB3 indicating their preference for the sn-2 position of glycerophospholipids. The thin layer chromatography analysis of the phospholipid classes after incubation of epidermal cells with [14C]-labeled HxB3, TrXB3, 12-hydroxy-eicosatetraenoic acid (12-HETE), 12-oxo-ETE, or 15-HETE showed that 12-HETE was the most esterified (12-HETE > 15-HETE > TrXB3 > 12-oxo-ETE > HxB3). HxB3 and TrXB3 were mainly esterified in phosphatidyl-choline and phosphatidyl-ethanolamine. HxB3 was also enzymatically converted into TrXB3 in vitro. HxB3 epoxide hydrolase-like activity was not observed when boiled tissue was incubated with [14C]-HxB3, this activity being located in the cytosol fraction (100,000 × g supernatant) of fresh tissue. These findings suggest that in vivo some part of HxB3 is transformed into TrXB3 and both compounds are partially incorporated into the phospholipids. arachidonic acid hydroperoxyeicosatetraenoic acid hepoxilin A3, 8-hydroxy-11,12-epoxy-5,9,14-eicosatrienoic acid hepoxilin B3, 10-hydroxy-11,12-epoxy-5,8,14-eicosatrienoic acid lipoxygenase hydrogenated methyl ester trimethylsilyl ether methyl ester trimethylsilyl ether 12-oxo-eicosatetraenoic acid phosphatidyl-choline phosphatidyl-ethanolamine phosphatidyl-inositol phosphatidyl-serine reverse phase-high performance liquid chromatography sphingomyelin trioxilin A3, 8,11,12-trihydroxy-5,9,14-eicosatrienoic acid trioxilin B3, 10,11,12-trihydroxy-5,8,14-eicosatrienoic acid 12-Lipoxygenase (12-LO) is the major arachidonic acid (AA) oxygenation pathway in epidermal cells with total product formation generally exceeding cyclooxygenase activity (Holtzman et al., 1989Holtzman M.J. Turk J. Pentland A. A regiospecific monooxygenase with novel stereopreference is the major pathway for arachidonic acid oxygenation in isolated epidermal cells.J Clin Invest. 1989; 84: 1446-1453Crossref PubMed Scopus (51) Google Scholar;Solá et al., 1992Solá J. Godessart N. Vila L. Puig L. de Moragas J.M. Epidermal cell-polymorphonuclear leucocyte cooperation in the formation of leukotriene B4 by transcellular biosynthesis.J Invest Dermatol. 1992; 98: 333-339Crossref PubMed Scopus (51) Google Scholar). Platelet-type 12-LO has been found to be the predominant isoenzyme expressed in human and murine skin epidermis (Takahashi et al., 1993Takahashi Y. Ramesh Reddy G. Ueda N. Yamamoto S. Arase S. Arachidonate 12-lipoxygenase of platelet-type in human epidermal cells.J Biol Chem. 1993; 268: 16443-16448Abstract Full Text PDF PubMed Google Scholar;Hussain et al., 1994Hussain H. Shornick L.P. Shannon V.R. Wilson J.D. Funk C.D. Pentland A.P. Holtzman M.J. Epidermis contains platelet-type 12-lipoxygenase that is overexpressed in germinal layer keratinocytes in psoriasis.Am J Physiol. 1994; 266: C243-C253PubMed Google Scholar;Krieg et al., 1995Krieg P. Kinzig A. Ress-Löschke M. et al.12-Lipoxygenase isoenzymes in mouse skin tumor development.Mol Carcinog. 1995; 14: 118-129Crossref PubMed Scopus (65) Google Scholar) and an “epidermal”-type 12-LO that functionally resembles the platelet-type 12-LO is also present in murine epidermis (Van Dijk et al., 1995Van Dijk K.W. Steketee K. Havekes L. Frants R. Hofker M. Genomic and cDNA cloning of a novel mouse lipoxygenase gene.Biochim Biophys Acta. 1995; 1259: 4-8Crossref PubMed Scopus (23) Google Scholar;Funk et al., 1996Funk C.D. Keeney D.S. Oliw E.H. Boeglin W.E. Brash A.R. Functional expression and cellular localization of a mouse epidermal lipoxygenase.J Biol Chem. 1996; 271: 23338-23344Crossref PubMed Scopus (81) Google Scholar;Kinzig et al., 1997Kinzig A. Fürstenberger G. Bürger F. et al.Murine epidermal lipoxygenase (Aloxe) encodes a 12-lipoxygenase isoform.FEBS Lett. 1997; 402: 162-166Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). We previously reported that, in addition to 12-hydroxyeicosatetraenoic acid (12-HETE), normal human epidermis incubated with exogenous AA produces 12-oxo-eicosatetraenoic acid (12-oxo-ETE), hepoxilin A3 (HxA3), and hepoxilin B3 (HxB3) through the 12-LO pathway (Antón et al., 1995Antón R. Abián J. Vila L. Characterization of arachidonic acid metabolites through the 12-lipoxygenase pathway in human epidermis by high-performance liquid chromatography and gas chromatography/mass spectrometry.in: J Mass Spectrom Rapid Commun Mass Spectrom. 1995: S169-S182Google Scholar;Antón and Vila, 2000Antón R. Vila L. Stereoselective biosynthesis of hepoxilin B3 in human epidermis.J Invest Dermatol. 2000; 114: 554-559https://doi.org/10.1046/j.1523-1747.2000.00903.xCrossref PubMed Scopus (24) Google Scholar). Recently, we also observed increased levels of nonesterified hepoxilins and trioxilins in the psoriatic scales (Antón et al., 1998Antón R. Puig L. Esgleyes T. de Moragas J.M. Vila L. Occurrence of hepoxilins and trioxilins in psoriatic lesions.J Invest Dermatol. 1998; 110: 303-310https://doi.org/10.1046/j.1523-1747.1998.00159.xCrossref PubMed Scopus (28) Google Scholar). Normal human epidermis synthesized only one of the two possible 10-hydroxy epimers of HxB3 whose formation is probably catalyzed by 12-LO (Antón et al., 1995Antón R. Abián J. Vila L. Characterization of arachidonic acid metabolites through the 12-lipoxygenase pathway in human epidermis by high-performance liquid chromatography and gas chromatography/mass spectrometry.in: J Mass Spectrom Rapid Commun Mass Spectrom. 1995: S169-S182Google Scholar;Antón and Vila, 2000Antón R. Vila L. Stereoselective biosynthesis of hepoxilin B3 in human epidermis.J Invest Dermatol. 2000; 114: 554-559https://doi.org/10.1046/j.1523-1747.2000.00903.xCrossref PubMed Scopus (24) Google Scholar). Hepoxilins exert action on plasma permeability on skin (Laneuville et al., 1991Laneuville O. Corey E.J. Couture R. Pace-Asciak C.R. Hepoxilin A3 increases vascular permeability in the rat skin.Eicosanoids. 1991; 4: 95-97PubMed Google Scholar;Wang et al., 1996Wang M.M. Demin P.M. Pace-Asciak C.R. Epimer-specific actions of hepoxilins A3 and B3 on PAF- and bradykinin-evoked vascular permeability in the rat skin in vivo.Adv Exp Med Biol. 1996; 416: 239-241Crossref PubMed Google Scholar,Wang et al., 1999aWang M.M. Demin P.M. Pace-Asciak C.R. Stereoselective actions of hepoxilins A3 and B3 and their cyclopropane analogs (HxDeltaA3 and HxDeltaB3) on bradykinin and PAF-evoked potentiation of vascular leakage in rat skin.Gen Pharmac. 1999; 33: 377-382Crossref PubMed Scopus (5) Google Scholar;Wang et al., 1999bWang M.M. Reynaud D. Pace-Asciak C.R. In vivo stimulation of 12(S)-lipoxygenase in the rat skin by bradykinin I and platelet activating factor: formation of 12(S)-HETE and hepoxilins, and actions on vascular permeability.Biochim Biophys Acta Lipids Lipid Metab. 1999; 1436: 354-362Crossref PubMed Scopus (27) Google Scholar), and induce a specific-receptor-dependent Ca2+ mobilization from endogenous sources (Dho et al., 1990Dho S. Grinstein S. Corey E.J. Su W.G. Pace-Asciak C.R. Hepoxilin A3 induces changes in cytosolic calcium, intracellular pH and membrane potential in human neutrophils.Biochem J. 1990; 266: 63-68Crossref PubMed Scopus (70) Google Scholar;Laneuville et al., 1993Laneuville O. Reynaud D. Grinstein S. Nigam S. Pace-Asciak C.R. Hepoxilin A3 inhibits the rise in free intracellular calcium evoked by formyl-methionyl-leucyl-phenylalanine, platelet-activating factor and leukotriene B4.Biochem J. 1993; 295: 393-397Crossref PubMed Scopus (36) Google Scholar) and the release of AA and diacylglycerol (Nigam et al., 1993Nigam S. Müller S. Pace-Asciak C.R. Hepoxilins activate phospholipase D in the human neutrophil.Dev Oncol. 1993; 71: 249-252Google Scholar). Interestingly, only the epimer 10(R)-HxB3, which is probably the epimer synthesized by normal epidermis (Antón et al., 1995Antón R. Abián J. Vila L. Characterization of arachidonic acid metabolites through the 12-lipoxygenase pathway in human epidermis by high-performance liquid chromatography and gas chromatography/mass spectrometry.in: J Mass Spectrom Rapid Commun Mass Spectrom. 1995: S169-S182Google Scholar;Antón and Vila, 2000Antón R. Vila L. Stereoselective biosynthesis of hepoxilin B3 in human epidermis.J Invest Dermatol. 2000; 114: 554-559https://doi.org/10.1046/j.1523-1747.2000.00903.xCrossref PubMed Scopus (24) Google Scholar), stereospecifically enhances the vascular permeability evoked by intradermal injection of the platelet-activating factor (Wang et al., 1996Wang M.M. Demin P.M. Pace-Asciak C.R. Epimer-specific actions of hepoxilins A3 and B3 on PAF- and bradykinin-evoked vascular permeability in the rat skin in vivo.Adv Exp Med Biol. 1996; 416: 239-241Crossref PubMed Google Scholar;Wang et al., 1999aWang M.M. Demin P.M. Pace-Asciak C.R. Stereoselective actions of hepoxilins A3 and B3 and their cyclopropane analogs (HxDeltaA3 and HxDeltaB3) on bradykinin and PAF-evoked potentiation of vascular leakage in rat skin.Gen Pharmac. 1999; 33: 377-382Crossref PubMed Scopus (5) Google Scholar;Wang et al., 1999bWang M.M. Reynaud D. Pace-Asciak C.R. In vivo stimulation of 12(S)-lipoxygenase in the rat skin by bradykinin I and platelet activating factor: formation of 12(S)-HETE and hepoxilins, and actions on vascular permeability.Biochim Biophys Acta Lipids Lipid Metab. 1999; 1436: 354-362Crossref PubMed Scopus (27) Google Scholar). On the other hand, less polar eicosanoids and octadecanoids, such as monohydroxy and epoxy derivatives of arachidonic and linoleic acids, have been found to esterify into cell phospholipids. Oxidized phospholipids have pro-inflammatory activities and are involved in atherogenesis, psoriasis, and other inflammatory diseases. In human epidermis, HxB3 can be further converted into trioxilin B3 (TrXB3, 10,11,12-trihydroxy-5,8,14-eicosatrienoic acid) (Antón et al., 1995Antón R. Abián J. Vila L. Characterization of arachidonic acid metabolites through the 12-lipoxygenase pathway in human epidermis by high-performance liquid chromatography and gas chromatography/mass spectrometry.in: J Mass Spectrom Rapid Commun Mass Spectrom. 1995: S169-S182Google Scholar). As relevant biologic activity for trioxilins has not been reported (Pace-Asciak et al., 1999Pace-Asciak C.R. Reynaud D. Demin P. Nigam S. The hepoxilins – a review.Adv Exp Med Biol. 1999; 447: 123-132Crossref PubMed Scopus (45) Google Scholar) these compounds could represent a pathway for hepoxilin inactivation. Enzymatic transformation of hepoxilins into trihydroxy compound was originally reported in rat lung (Pace-Asciak et al., 1983Pace-Asciak C.R. Granström E. Samuelsson B. Arachidonic acid epoxides. Isolation and structure of two hydroxy epoxide intermediates in the formation of 8,11,12- and 10,11,12-trihydroxyeicosatrienoic acids.J Biol Chem. 1983; 258: 6835-6840Abstract Full Text PDF PubMed Google Scholar). No data about the enzymatic metabolism of HxB3 in human epidermis are at present available, however. This prompted us to extend our previous investigations (Antón et al., 1995Antón R. Abián J. Vila L. Characterization of arachidonic acid metabolites through the 12-lipoxygenase pathway in human epidermis by high-performance liquid chromatography and gas chromatography/mass spectrometry.in: J Mass Spectrom Rapid Commun Mass Spectrom. 1995: S169-S182Google Scholar;Antón et al., 1998Antón R. Puig L. Esgleyes T. de Moragas J.M. Vila L. Occurrence of hepoxilins and trioxilins in psoriatic lesions.J Invest Dermatol. 1998; 110: 303-310https://doi.org/10.1046/j.1523-1747.1998.00159.xCrossref PubMed Scopus (28) Google Scholar;Antón and Vila, 2000Antón R. Vila L. Stereoselective biosynthesis of hepoxilin B3 in human epidermis.J Invest Dermatol. 2000; 114: 554-559https://doi.org/10.1046/j.1523-1747.2000.00903.xCrossref PubMed Scopus (24) Google Scholar) on the presence of HxB3 in the phospholipid fraction of psoriatic lesions, on HxB3 incorporation into the different phospholipid classes in human epidermal cells, and on the enzymatic pathways involved in the catabolism of HxB3. [1–14C]-AA (55–58 mCi per mmol) was supplied by Amersham Ibérica (Madrid, Spain). Phospholipase A2 from bee venom was supplied by Sigma-Aldrich Química (Madrid, Spain). Hydrated platinum (IV) oxide was purchased from ICN Biochemicals (Costa Mesa, CA). Hydrogen gas was purchased from Abelló Oxígeno-Linde (Barcelona, Spain). 1-Stearoyl-2-[1–14C]-arachidonoyl-L-3-phosphatidyl choline was obtained from Amersham Ibérica. (±)-HxB3 were from Cascade Biochem (Berkshire, U.K.). All high performance liquid chromatography (HPLC) solvents were supplied by Scharlau (Barcelona, Spain) and solvents for mass spectrometry analysis and N,O-bis-(trimethylsilyl)-trifluoroacetamide (BSTFA) were purchased from Merck (Darmstadt, Germany). Epidermis was isolated from normal skin, obtained from plastic surgery, using the Liu and Karasek technique (Liu and Karasek, 1978Liu S.C. Karasek M. Isolation and growth of adult human epidermal keratinocytes in cell culture.J Invest Dermatol. 1978; 71: 157-162Crossref PubMed Scopus (214) Google Scholar) with minor modifications (Solá et al., 1992Solá J. Godessart N. Vila L. Puig L. de Moragas J.M. Epidermal cell-polymorphonuclear leucocyte cooperation in the formation of leukotriene B4 by transcellular biosynthesis.J Invest Dermatol. 1992; 98: 333-339Crossref PubMed Scopus (51) Google Scholar). Briefly, narrow strips of skin were cut and rinsed twice in phosphate-buffered saline, pH 7.4, free of Ca2+ and Mg2+ (PBS*). The strips were then placed in PBS* containing 0.5% trypsin (wt/vol) (Difco Laboratories, Paisley, Scotland) and kept at 37°C for 50–60 min. When dermo-epidermal detachment occurred, epidermal strips were transferred to a culture medium [Dulbecco's modified Eagle's medium (DMEM) (Flow Laboratories, Irvine, Scotland) + 2 mM glutamine + 1 mM sodium pyruvate] containing 10% vol/vol fetal bovine serum (FBS) (Flow Laboratories). After peeling the epidermis from the dermis, the epidermal fragments were washed in DMEM. Epidermal fragments were isolated by filtration through a sterile gauze. The remaining sheets were divided into small fragments with a surgical blade (approximately 1 mm2) and used for experiments without further manipulation. To obtain epidermal cell suspensions, after peeling the epidermis from the dermis, the epidermal fragments were then gently stirred for 10–15 min in DMEM supplemented with 2 mM glutamine, 1 mM sodium pyruvate, and 10% vol/vol FBS. The cellular suspension thus obtained was filtered through a sterile gauze to discard fragments of whole epidermis and then kept at 37°C until incubation. Only cell suspensions with viability greater than 95% were used. Fragments of fresh human epidermis and epidermal cell suspensions were used immediately. Scales from untreated patients with chronic stable plaque psoriasis involving more than 10% of body surface were removed by scraping with a surgical blade and stored at -80°C under an N2 atmosphere until analysis. Psoriatic scales (500–700 mg) were cut and mechanically homogenized with a Turrax T-8 in 3 ml of degassed distilled water containing 0.025‰ (wt/vol) BHT placed in an ice-water bath under an N2 atmosphere. Next, 100,000 cpm of 1-stearoyl-2-([1–14C]-arachidonyl)-phosphatidylcholine were added as internal standard. Immediately, total lipids were extracted as described previously (Bligh and Dyer, 1959Bligh E.G. Dyer W.J. A rapid method of total lipid extraction and purification.Can J Biochem Physiol. 1959; 37: 911-917Crossref PubMed Scopus (40104) Google Scholar). Extracts of CHCl3 were dried under an N2 stream and redissolved in 200 µl of CHCl3. Phospholipids were separated from free fatty acids and neutral lipids as described previously (Kaluzny et al., 1985Kaluzny M.A. Duncan L.A. Merritt M.V. Epps D.E. Rapid separation of lipid classes in high yield and purity using bonded phase columns.J Lipid Res. 1985; 26: 135-140Abstract Full Text PDF PubMed Google Scholar) using solid phase extraction chromatography. All solvents were supplemented with 0.025‰ (wt/vol) BHT and the extraction was performed under an N2 atmosphere. The phospholipid fraction was divided in two aliquots and phospholipids were hydrolyzed by following two different procedures: alkaline saponification and treatment with purified phospholipase A2 (PLA2). For alkaline saponification phospholipid extracts were dried under an N2 stream, the residue was redissolved in 850 µl of MeOH/CHCl3 8:1, and 150 µl 40% wt/vol KOH were added. The mixture was allowed to react for 30 min at 60°C under an N2 atmosphere (Kühn et al., 1994Kühn H. Belkner J. Suzuki H. Yamamoto S. Oxidative modification of human lipoproteins by lipoxygenases of different positional specificities.J Lipid Res. 1994; 35: 1749-1759Abstract Full Text PDF PubMed Google Scholar). The reaction was stopped by adding 700 µl of 50 mM phosphate buffer pH = 7.4 and acidifying until pH 2–3. Free fatty acids were then extracted twice with 2 ml diethyl ether/hexane 1:1. The reaction yield measuring release of [1–14C]-AA from 1-stearoyl-2-[1–14C]-arachidonoyl-L-3-phosphatidylcholine was about 94%. For specific hydrolysis of the sn-2 position, phospholipid extracts were dried under an N2 stream, and the residue was redissolved in 10 µl of CHCl3 and 70 µl of 30 mM borate buffer (pH = 9.0) supplemented with 1.6 mM CaCl2. The mixture was then shaken vigorously and CHCl3 was removed under an N2 stream. 850 U of bee venom PLA2 in 85 µl of 30 mM borate buffer pH 9.0 were added and the mixture was allowed to react for 45 min at 37°C under an N2 atmosphere with continuous agitation. Afterwards, another 850 U of PLA2 were added and allowed to react for another 100 min (Smiley et al., 1991Smiley P.L. Stremler K.E. Prescott S.M. Zimmerman G.A. McIntyre T.M. Oxidatively fragmented phosphatidylcholines activate human neutrophils through the receptor for platelet-activating factor.J Biol Chem. 1991; 266: 11104-11110Abstract Full Text PDF PubMed Google Scholar). Reaction was stopped by acidification until pH 2–3 and lipids were extracted as described byBligh and Dyer, 1959Bligh E.G. Dyer W.J. A rapid method of total lipid extraction and purification.Can J Biochem Physiol. 1959; 37: 911-917Crossref PubMed Scopus (40104) Google Scholar. After analysis of the free [14C]-AA released from 1-stearoyl-2-[1–14C]-arachidonoyl-L-3-phosphatidylcholine the yield of the reaction was about 96%. Labeled 12-LO-derived eicosanoids were obtained from incubations of fragments of human epidermis, previously treated with 200 µM aspirin for 15 min, with 100 µM [14C]-AA for 30 min at 37°C, and processed as described previously (Antón and Vila, 2000Antón R. Vila L. Stereoselective biosynthesis of hepoxilin B3 in human epidermis.J Invest Dermatol. 2000; 114: 554-559https://doi.org/10.1046/j.1523-1747.2000.00903.xCrossref PubMed Scopus (24) Google Scholar). Supernatants of several incubations were mixed and processed together. Supernatants of the incubations, which had a ratio MeOH:H2O of 1:1 and a pH of 2–3, were extracted three times with half a volume of diethyl ether:hexane 1:1. Extracts were evaporated under an N2 stream until dryness, redissolved in the eluent of reverse phase high performance liquid chromatography (RP-HPLC), and chromatographied as described later. Fractions containing hepoxilins (14–20 min) and 12-oxo-ETE plus 12-HETE (25–33 min) were collected and purified by straight-phase HPLC as reported previously (Antón et al., 1995Antón R. Abián J. Vila L. Characterization of arachidonic acid metabolites through the 12-lipoxygenase pathway in human epidermis by high-performance liquid chromatography and gas chromatography/mass spectrometry.in: J Mass Spectrom Rapid Commun Mass Spectrom. 1995: S169-S182Google Scholar). The specific activity of the labeled compounds was assumed to be the same as the [14C]-AA batch used in their preparation as AA is labeled in the C of the carboxyl group, which is conserved in all these compounds. Labeled TrXB3 was obtained from incubations of fragments of human epidermis with 10 µM [14C]-labeled HxB3 for 30 min at 37°C. After the incubation period one volume of methanol and 1 M HCl to yield a pH of 2–3 were added. Supernatants of several incubations were mixed and processed together. These supernatants were then extracted three times with half a volume of diethyl ether:hexane 1:1. Extracts were evaporated under an N2 stream until dryness, redissolved in the eluent of RP-HPLC, and chromatographied as described above. The fraction containing TrXB3 (4–11 min) was collected and purified by straight-phase HPLC as reported previously (Antón et al., 1995Antón R. Abián J. Vila L. Characterization of arachidonic acid metabolites through the 12-lipoxygenase pathway in human epidermis by high-performance liquid chromatography and gas chromatography/mass spectrometry.in: J Mass Spectrom Rapid Commun Mass Spectrom. 1995: S169-S182Google Scholar). The specific activity of the labeled TrXB3 was assumed to be the same as that of [14C]-HxB3. [14C]-15-hydroperoxyeicosatetraenoic acid (15-HPETE) was obtained by incubating [14C]-AA with soybean LO and was purified by straight-phase HPLC, after reduction with NaBH4, as previously described (Camacho et al., 1995Camacho M. Godessart N. Antón R. García M. Vila L. Interleukin-1 enhances the ability of cultured umbilical vein endothelial cells to oxidize linoleic acid.J Biol Chem. 1995; 270: 17279-17286Crossref PubMed Scopus (52) Google Scholar). The specific activity of the labeled 15-HETE was assumed to be the same as that of the [14C]-AA batch used in its preparation. Epidermis fragments (1–2 g) were homogenized and microsomal (100,000 × g pellet) and cytosolic (100,000 × g supernatant) fractions were obtained as described previously (Antón and Vila, 2000Antón R. Vila L. Stereoselective biosynthesis of hepoxilin B3 in human epidermis.J Invest Dermatol. 2000; 114: 554-559https://doi.org/10.1046/j.1523-1747.2000.00903.xCrossref PubMed Scopus (24) Google Scholar) and incubated with 10 µM [14C]-HxB3 or unlabeled HxB3 (as required) at 37°C for 30 min. Eicosanoids were analyzed by RP-HPLC, and by gas chromatography-mass spectrometry (GC-MS) when necessary, as described below. Ten million epidermal cells in suspension were incubated in 0.5 ml of RPMI-1640 containing 10 µM of [14C]-15-HETE, [14C]-12-HETE, [14C]-12-oxo-ETE, [14C]-HxB3, or [14C]-TrXB3 for 4 h at 37°C. Reactions were stopped by adding a volume of a cold solution of 2% acetic acid in methanol. Cells were then centrifuged and supernatants were stored at -80°C for further analysis. Pellets were suspended in 1.5 ml of 2% acetic acid in methanol followed by 1.5 ml of water. Lipids were extracted according to the method described byBligh and Dyer, 1959Bligh E.G. Dyer W.J. A rapid method of total lipid extraction and purification.Can J Biochem Physiol. 1959; 37: 911-917Crossref PubMed Scopus (40104) Google Scholar. Extracts were dried under an N2 stream and the residues were redissolved in 35 µl of methanol:chloroform 1:2. Labeled phospholipids were analyzed as previously described (Godessart et al., 1996Godessart N. Camacho M. López-Belmonte J. Antón R. García M. de Moragas J.-M. Vila L. Prostaglandin H-synthase-2 is the main enzyme involved in the biosynthesis of octadecanoids from linoleic acid in human dermal fibroblasts stimulated with IL-1β.J Invest Dermatol. 1996; 107: 726-732Crossref PubMed Scopus (26) Google Scholar). Chromatography was performed as previously described (Antón et al., 1998Antón R. Puig L. Esgleyes T. de Moragas J.M. Vila L. Occurrence of hepoxilins and trioxilins in psoriatic lesions.J Invest Dermatol. 1998; 110: 303-310https://doi.org/10.1046/j.1523-1747.1998.00159.xCrossref PubMed Scopus (28) Google Scholar). Quantitative analysis of 12-LO-derived compounds was done by injecting the samples directly into the column without further manipulation. The column was then coupled on line with a radioactivity detector (Beckman-171) equipped with a liquid scintillation cell. Eluents were mixed with scintillation cocktail pumped at 3 ml per min. Data from the detector were processed with a System Gold Software Beckman in a PC computer. When isolation of eluted material was required, fractions were collected either on exit from the radioactivity detector equipped with a solid scintillation cell, or from the column, depending on labeled or unlabeled samples. In this way we collected fractions of TrXs at 4–11 min, Hxs at 14–20 min, HODEs at 20–25 min, 12-oxo-ETE (and also HODEs and 15-HETE) at 25–27 min, and HETEs 27–40 min. These fractions were later concentrated by liquid-liquid extraction after adjusting the MeOH:H2O ratio to 1:1. Dried extracts were then derivatized for GC-MS analysis. Moreover, [1–14C]-AA from an internal standard was collected in order to assess quantitative ester hydrolysis and counted in a liquid scintillation counter. Both kinds of hydrolysis were quantitative. All fractions except that of 12-oxo-ETE were derivatized to their hydrogenated methyl ester trimethylsilyl ethers (ME-H-TMS) as described previously (Antón et al., 1998Antón R. Puig L. Esgleyes T. de Moragas J.M. Vila L. Occurrence of hepoxilins and trioxilins in psoriatic lesions.J Invest Dermatol. 1998; 110: 303-310https://doi.org/10.1046/j.1523-1747.1998.00159.xCrossref PubMed Scopus (28) Google Scholar). 12-oxo-ETE was first transformed into its methyl ester trimethylsilyl ether (ME-TMS), to demonstrate it was not contaminated with 12-HETE, before transformation to its ME-H-TMS derivative. All samples were dried and redissolved in heptane/BSTFA. Analysis by GC-MS was performed as previously described (Antón et al., 1995Antón R. Abián J. Vila L. Characterization of arachidonic acid metabolites through the 12-lipoxygenase pathway in human epidermis by high-performance liquid chromatography and gas chromatography/mass spectrometry.in: J Mass Spectrom Rapid Commun Mass Spectrom. 1995: S169-S182Google Scholar,Antón et al., 1998Antón R. Puig L. Esgleyes T. de Moragas J.M. Vila L. Occurrence of hepoxilins and trioxilins in psoriatic lesions.J Invest Dermatol. 1998; 110: 303-310https://doi.org/10.1046/j.1523-1747.1998.00159.xCrossref PubMed Scopus (28) Google Scholar). In this case, the GC column was a TRB-1 fused silica capillary column (15 m length, 0.25 mm internal diameter, 0.25 µm film thickness, Tracer Analítica, Barcelona, Spain). The gas chromatograph and mass spectrometer were Hewlett-Packard 6890 series and 5973 model, respectively. After isolation, the phospholipids were hydrolyzed by two procedures: treatment with purified PLA2 or with KOH. Samples were then subjected to HPLC fractionation. Hx and TrX fractions were then derivatized and subjected to GC-MS analysis using the full scan mode. Monitoring suitable ions for HxB3 (m/z 73, 269, 282, and 311), the ME-TMS derivatives of the Hxs fraction from psoriatic scales subjected to GC-MS analysis eluted as a single peak. The EI mass spectrum was consistent with the structure of 10-hydroxy-11,12-epoxy-5,8,14-eicosatrienoic acid (HxB3) and it was essentially identical to that obtained with authentic (±)HxB3. (See our previous studies for details of mass spectra (Antón et al., 1995Antón R. Abián J. Vila L. Characterization of arachidonic acid metabolites through the 12-lipoxygenase pathway in human epidermis by high-performan" @default.
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• W2074616170 title "Hepoxilin B3 and its Enzymatically Formed Derivative Trioxilin B3 are Incorporated into Phospholipids in Psoriatic Lesions" @default.
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