Stereoselective biosynthesis of hepoxilin B3 in human epidermis
Stereoselective biosynthesis of hepoxilin B3 in human epidermis
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DOI：
10.1046/j.1523-1747.2000.00903.x
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发表时间：
2000-03-01
影响因子：
6.5
通讯作者：
Vila, L
中科院分区：
文献类型：
--
作者：
Antón, R;Vila, L
我们以前报道过正常人表皮形成12-氧代-二十碳四烯酸和肝氧素B-3作为主要的类二十烷酸，并且肝氧素和trioxilins在银屑病病变中显著升高。我们还观察到，正常表皮仅合成两种可能的10-羟基差向异构体的hepoxilin B-3，这表明其酶的起源。本研究探讨了在人表皮中参与形成肝氧素B-3的酶途径。将人表皮碎片或细胞级分与[C-14]-花生四烯酸或真正的12（S）-氢过氧二十碳四烯酸一起孵育。通过高效液相色谱法、气相色谱-质谱法或这两种技术的组合来分析产物。七叶壳和去甲二氢愈创木酸以浓度依赖性方式抑制hepoxilin B-3、12-oxo-eicosatetraenoic acid、trioxilins和12-hydroxyeicosatetraenoic acid的形成。12-脂氧合酶活性主要位于微粒体组分（100，000 x g颗粒）中，并形成12-羟基二十碳四烯酸、肝氧素B-3和12-氧代-二十碳四烯酸。在与真正的12（S）-氢过氧二十碳四烯酸孵育的亚细胞组分中未观察到肝氧素B-3合成活性，尽管当与花生四烯酸孵育时，它至少位于微粒体组分中。使用重组血小板型12-脂氧合酶的制剂获得了类似的结果，当与花生四烯酸孵育时，除了12-羟基二十碳四烯酸外，该重组血小板型12-脂氧合酶还产生12-氧代-二十碳四烯酸和肝氧素B-3，但当与12-氢过氧二十碳四烯酸孵育时则没有。然而，重组12-脂氧合酶产生的12-氧代-二十碳四烯酸和肝氧素B-3-12-羟基二十碳四烯酸的比例低于表皮。我们的研究结果支持的概念，12-脂氧合酶催化形成的肝氧素B-3和12-氧代-二十碳四烯酸。
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.