Du Liping, Yermalitsky Valery, Hachey David L, Jagadeesh Setti G, Falck John R, Keeney Diane S
Department of Medicine/Dermatology and Biochemistry, Vanderbilt University, 607 Light Hall (0146), Nashville, TN 37232-0146, USA.
J Pharmacol Exp Ther. 2006 Jan;316(1):371-9. doi: 10.1124/jpet.105.093922. Epub 2005 Sep 16.
The epidermis expresses cyclooxygenases, lipoxygenases, and cytochromes P450, which utilize arachidonic acid to generate a diverse array of lipid mediators affecting epidermal cellular differentiation and functions. Recent studies show that mouse epidermis expresses CYP2B19, a keratinocyte-specific epoxygenase that generates 11,12- and 14,15-epoxyeicosatrienoic (EET) acids from arachidonate. We studied CYP2B19-dependent metabolism in mouse epidermal microsomes, reconstituted in the presence of [1-(14)C]arachidonic acid. The majority of the (14)C products formed independently of NADPH, indicative of robust epidermal cyclooxygenase and lipoxygenase activities. We studied two NADPH-dependent products generated in a highly reproducible manner from arachidonate. One of these (product I) coeluted with the CYP2B19 product 14,15-EET on a reversed-phase high-performance liquid chromatography (HPLC) system; there was no evidence for other regioisomeric EET products. Further analyses proved that product I was not an epoxy fatty acid, based on different retention times on a normal-phase HPLC system and failure of product I to undergo hydrolysis in acidic solution. We analyzed purified epidermal (14)C products by liquid chromatography negative electrospray ionization mass spectrometry. Structures of the NADPH-dependent products were confirmed to be 12-oxo-5,8,14-eicosatrienoic acid (I) and 12-hydroxy-5,8,14-eicosatrienoic acid (II). This was the first evidence for a 12-hydroxy-5,8,14-eicosatrienoic acid biosynthetic pathway in mouse epidermis. Epidermal microsomes also generated 12-hydroperoxy, 12-hydroxy, and 12-oxo eicosatetraenoic acids from arachidonate, possible intermediates in the 12-hydroxy-5,8,14-eicosatrienoic acid biosynthetic pathway. These results predict that hydroxyeicosatrienoic acids are synthesized from arachidonate in human epidermis. This would have important implications for human skin diseases given the known pro- and anti-inflammatory activities of stereo- and regioisomeric hydroxyeicosatrienoic acids.
表皮表达环氧化酶、脂氧化酶和细胞色素P450，它们利用花生四烯酸生成一系列影响表皮细胞分化和功能的脂质介质。最近的研究表明，小鼠表皮表达CYP2B19，一种角质形成细胞特异性环氧化酶，可从花生四烯酸生成11,12-和14,15-环氧二十碳三烯酸（EET）。我们在存在[1-(14)C]花生四烯酸的情况下重组了小鼠表皮微粒体，研究了CYP2B19依赖性代谢。大多数(14)C产物的形成不依赖于NADPH，这表明表皮环氧化酶和脂氧化酶活性很强。我们研究了从花生四烯酸以高度可重复的方式产生的两种NADPH依赖性产物。其中一种（产物I）在反相高效液相色谱（HPLC）系统上与CYP2B19产物14,15-EET共洗脱；没有证据表明存在其他区域异构体EET产物。进一步分析证明，基于在正相HPLC系统上不同的保留时间以及产物I在酸性溶液中不发生水解，产物I不是环氧脂肪酸。我们通过液相色谱负电喷雾电离质谱分析了纯化的表皮(14)C产物。NADPH依赖性产物的结构被确认为12-氧代-5,8,14-二十碳三烯酸（I）和12-羟基-5,8,14-二十碳三烯酸（II）。这是小鼠表皮中12-羟基-5,8,14-二十碳三烯酸生物合成途径的首个证据。表皮微粒体还从花生四烯酸生成12-氢过氧、12-羟基和12-氧代二十碳四烯酸，它们可能是12-羟基-5,8,14-二十碳三烯酸生物合成途径中的中间体。这些结果预测，人表皮中花生四烯酸可合成羟基二十碳三烯酸。鉴于立体和区域异构体羟基二十碳三烯酸已知的促炎和抗炎活性，这对人类皮肤疾病具有重要意义。
