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Identification of 5-Keto-(7E,9E,11Z,14Z)-Eicosatetraenoic Acid as a Novel Nonenzymatic Rearrangement Product of Leukotriene A4

https://doi.org/10.1006/abbi.1993.1539Get rights and content

Abstract

Leukotriene A4 (LTA4), the reaction product of 5-lipoxygenase in human polymorphonuclear (PMN) leukocytes, is transformed both to LTB4 and a mixture of 5,6- and 5,12-dihydroxy-eicosatetraenoic acids (diHETE) via nonenzymatic hydrolysis. Evidence has been obtained that LTA4 is also converted to 5-keto-(7E,9E,11Z,14Z)-eicosatetraenoic acid (5-oxo-ETE). The compound was isolated from the products of the 5-lipoxygenase reaction and its structure elucidated by UV spectroscopy, LC-MS, two-dimensional [1H]NMR spectroscopy and chemical reduction to the corresponding alcohol. The 5-oxo-ETE represented about 14% of the LTA4 hydrolysis products as compared to 72 and 14% for the 5,12-diHETE and 5,6-diHETE, respectively. A similar profile of hydrolysis products was obtained after incubation of synthetic LTA4 in aqueous buffer. Human PMN leukocytes produced 5-oxo-ETE in an arachidonic acid-dependent and MK-886-inhibitable manner. The 5-oxo-ETE caused 50% inhibition of 5-lipoxygenase activity at 1 μM. These results demonstrate that the noneazymatic conversion of LTA4, in addition to the previously described hydrolysis products, yields 5-oxo-ETE during both the 5-lipoxygenase reaction and arachidonic acid oxidation by human PMN leukocytes. They indicate that allylic epoxides can rearrange in aqueous media at physiological pH to spontaneously form β,γ-unsaturated ketones.

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    This phenomenon was reported for the injection of 12-HpETE or 15-HpETE onto an HPLC column without prior reduction [31]. However, the measured product might also be 5-oxo-7E,9E,11Z,14Z-eicosatetraenoic acid, a further nonenzymatic hydrolysis product of LTA4 [25], differing only in the position of the double bonds. Gravel et al. described an MS/MS fragment with m/z 203 Da for 5-oxo-7E,9E,11Z,14Z-eicosatetraenoic acid [25], which is also used for the applied LC–ESI–MS/MS analysis of 5-oxo-ETE.

  • A one-step method of 10,17-dihydro(pero)xydocosahexa-4Z,7Z,11E,13Z,15E,19Z- enoic acid synthesis by soybean lipoxygenase

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    The spectrum lacked a proton resonance with δ 6.15–6.24 ppm, which would have been present if the compound had a conjugated 11E,13E or 13E,15E fragment. Such resonances have been reported for several compounds with conjugated E,E double bonds, for example, all-trans isomers of 9(S)- and 13(S)-hydroxy linoleyl alcohols (14), 9(S)- and 13(S)-hydroxy monolinoleoyl glycerols (15), 5,6-dihydroxyeicosapenta-7E,9E,11Z,14Z,17Z-enoic acid (16), 5-ketoeicosatetra-7E,9E,11Z,14Z-enoic acid (17), 14,15-dihydroxyeicosatetra-5Z,8Z,10E,12E-enoic acid (18), and 5,12(S)-dihydroxyeicosatetra-6E,8E,10E-enoic acid (19). Only three types of protons that belonged to the 11E,13Z,15E fragment were observed in the NMR experiments (Table 1, Figs. 1, 3, 4).

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