Stereoselective biosynthesis of hepoxilin B3 in human epidermis

We previously reported that normal human epidermis forms 12-oxo-eicosatetraenoic acid and hepoxilin B-3 as major eicosanoids and that hepoxilins and trioxilins are dramatically elevated in psoriatic lesions. We also observed that normal epidermis only synthesized one of the two possible 10-hydroxy- epimers of hepoxilin B-3, suggesting its enzymatic origin. This study investigated the enzymatic pathways involved in the formation of hepoxilin B-3 in human epidermis. Human epidermal fragments or cell fractions were incubated with [C-14]-arachidonic acid or authentic 12(S)-hydroperoxyeicosatetraenoic acid. Products were analyzed by high-performance liquid chromatography, gas chromatography-mass spectrometry or a combination of both techniques. Esculetin and nordihydroguaiaretic acid inhibited formation of hepoxilin B-3, 12-oxo-eicosatetraenoic acid, trioxilins, and 12-hydroxyeicosatetraenoic acid in a concentration-dependent manner. 12-Lipoxygenase activity was mainly located in the microsomal fraction (100,000 x g pellet) and 12-hydroxyeicosatetraenoic acid, hepoxilin B-3, and 12-oxo-eicosatetraenoic acid were formed. The hepoxilin B-3-synthesizing activity was not observed in subcellular fractions incubated with authentic 12(S)-hydroperoxyeicosatetraenoic acid, although it was located at least in the microsomal fraction when incubated with arachidonic acid. Similar results were obtained using preparations of recombinant platelet-type 12-lipoxygenase that yielded 12-oxo-eicosatetraenoic acid and hepoxilin B-3 in addition to 12-hydroxyeicosatetraenoic acid, when incubated with arachidonic acid but not when incubated with 12-hydroperoxyeicosatetraenoic acid. Nevertheless, recombinant 12-lipoxygenase produced a lower ratio of 12-oxo-eicosatetraenoic acid and hepoxilin B-3-12-hydroxyeicosatetraenoic acid than epidermis. Our results support the concept that 12-lipoxygenase catalyzes the formation of hepoxilin B-3 and 12-oxo-eicosatetraenoic acid.