Formation of 14,15-hepoxilins of the A(3) and B(3) series through a 15-lipoxygenase and hydroperoxide isomerase present in garlic roots.
We report herein for the first time the formation by freshly grown garlic roots and the structural characterization of 14,15-epoxide positional analogs of the hepoxilins formed via the 15-lipoxygenase-induced oxygenation of arachidonic acid. These compounds are formed through the combined actions of a 15(S)-lipoxygenase and a hydroperoxyeicosatetraenoic acid (HPETE) isomerase. The compounds were formed when either arachidonic acid or 15-HPETE were used as substrates. Both the "A"-type and the "B"-type products are formed although the B-type compounds are formed in greater relative quantities. Chiral phase high performance liquid chromatography analysis confirmed the formation of hepoxilins from 15(S)- but not 15(R)-HPETE, indicating high stereoselectivity of the isomerase. Additionally, the lipoxygenase was of the 15(S)-type as only 15(S)-hydroxyeicosatetraenoic acid was formed when arachidonic acid was used as substrate. The structures of the products were confirmed by gas chromatography-mass spectrometry of the methyl ester trimethylsilyl ether derivatives as well as after characteristic epoxide ring opening catalytically with hydrogen leading to dihydroxy products. That 15(S)-lipoxygenase activity is of functional importance in garlic was shown by the inhibition of root growth by BW 755C, a dual cyclooxygenase/lipoxygenase inhibitor and nordihydroguaiaretic acid, a lipoxygenase inhibitor. Additional biological studies were carried out with the purified intact 14(S), 15(S)-hepoxilins, which were investigated for hepoxilin-like actions in causing the release of intracellular calcium in human neutrophils. The 14,15-hepoxilins dose-dependently caused a rise in cytosolic calcium, but their actions were 5-10-fold less active than 11(S), 12(S)-hepoxilins derived from 12(S)-HPETE. These studies provide evidence that 15(S)-lipoxygenase is functionally important to normal root growth and that HPETE isomerization into the hepoxilin-like structure may be ubiquitous; the hepoxilin-evoked release of calcium in human neutrophils, which is receptor-mediated, is sensitive to the location within the molecule of the hydroxyepoxide functionality.
Reynaud
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Research Institute, The Hospital for Sick Children, Toronto, Canada M5G 1X8.
Ali
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Demin
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Pace-Asciak
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D015126:8,11,14-Eicosatrienoic Acid / Q000031:analogs & derivatives* / Q000378:metabolism; D001093:Arachidonate 15-Lipoxygenase / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D005737:Garlic / Q000201:enzymology*; D006801:Humans; D019746:Intramolecular Oxidoreductases / Q000378:metabolism*; D009504:Neutrophils / Q000378:metabolism; D018517:Plant Roots / Q000201:enzymology; D010946:Plants, Medicinal; D011956:Receptors, Cell Surface / Q000378:metabolism
D011956:Receptors, Cell Surface; C061163:11-glutathionylhepoxilin A3; C022948:10-hydroxy-11,12-epoxyeicosa-5,8,14-trienoic acid; D001093:Arachidonate 15-Lipoxygenase; D019746:Intramolecular Oxidoreductases; C021377:hydroperoxide isomerase; D015126:8,11,14-Eicosatrienoic Acid