Model for metabolism of arachidonic acid by 5-lipoxygenase as 5, 6-LTA4 synthase in the sheep uterus: Evidence from in vitro studies

Abstract

Sheep uterine tissue is rich in the enzyme 5-lipoxygenase (5-LOX) that generates 5-hydroperoxyeicosatetraenoic acid (5-HPETE) on incubation with arachidonic acid (AA). The present study focuses on the ability of the enzyme 5-LOX to further act as 5, 6 leukotriene A4 (LTA 4 ) synthase using 5-HPETE as a substrate. On incubation of 5-LOX with 5-HPETE and analysis of products on reversed-phase high-performance liquid chromatography, a prominent peak with a characteristic conjugated triene peak having absorption maxima of 261, 271, and 281 nm appeared. Based on cochromatography, the peak was identified as 5(S), 6(S)-dihydroxyeicosatetraenoic acid (5[S], 6[S]-diHETE), the non-enzymatic product of 5, 6 LTA 4 . As the synthesis of 5,6-LTA 4 from 5-HPETE requires 8-LOX activity, the present study demonstrates the dual LOX, 5- and 8-LOX activity for uterine 5-LOX, that is, 5-,6- LTA 4 synthase in sheep uterus. In addition, endogenous 5,6-LTC 4 , the glutathione conjugate of 5,6-LTA 4 , was also observed in the sheep uterine homogenate, further demonstrating the synthesis of 5,6-LTA 4 in the sheep uterine tissue.