Fuchs J, Mehlhorn R J, Packer L
Department of Physiology and Anatomy, University of California, Berkeley.
J Invest Dermatol. 1989 Nov;93(5):633-40. doi: 10.1111/1523-1747.ep12319780.
Scavenging mechanisms for persistent free radicals were investigated using nitroxide-type radicals as model compounds. The free radical reducing activity of a) isolated thioredoxin reductase, a flavin containing oxidoreductase, b) skin homogenates, and c) the epidermis of hairless mice was studied by electron spin resonance spectroscopy. In all three systems, reduction rates of different classes of nitroxide free radicals exhibited the following order: oxazolidinoxy greater than piperidinoxy greater than dihydropyrroloxy. The main reductant for piperidinoxy radicals in mouse skin homogenate is ascorbic acid. Other reducing activities were stimulated by NAD(P)H and could be inhibited by N-ethyl maleimide, suggesting involvement of thiol-dependent processes. Mammalian thioredoxin, a competitive inhibitor of nitroxide reduction by thioredoxin reductase, significantly stimulates nitroxide scavenging in skin homogenate. Thioredoxin reductase did not significantly participate in nitroxide reduction in skin homogenates. At the surface of mouse epidermis a cationic dihydropyrroloxy nitroxide, which was stable in the presence of mammalian thioredoxin reductase was readily reduced. The epidermal reduction was inhibited by zinc, N-ethyl maleimide, and by heat (70 degrees C, 5 min). At least for mouse epidermis, reduction of a variety of nitroxides is a complex phenomenon involving enzymatic and nonenzymatic mechanisms and cannot be used as a specific assay for an enzyme, e.g., thioredoxin reductase. The study indicates the epidermis contains an effective antioxidant system that scavenges ascorbate-sensitive piperidinoxy nitroxides as well as more reducing radicals exemplified by dihydropyrroloxy nitroxides.
以氮氧自由基型自由基作为模型化合物，研究了持久性自由基的清除机制。通过电子自旋共振光谱法研究了以下物质的自由基还原活性：a）分离出的硫氧还蛋白还原酶，一种含黄素的氧化还原酶；b）皮肤匀浆；c）无毛小鼠的表皮。在所有这三个系统中，不同类别的氮氧自由基的还原速率呈现出以下顺序：恶唑烷氧基＞哌啶氧基＞二氢吡咯氧基。小鼠皮肤匀浆中哌啶氧基自由基的主要还原剂是抗坏血酸。其他还原活性受到NAD(P)H的刺激，并且可被N-乙基马来酰亚胺抑制，这表明涉及硫醇依赖性过程。哺乳动物硫氧还蛋白是硫氧还蛋白还原酶还原氮氧化物的竞争性抑制剂，它能显著刺激皮肤匀浆中的氮氧化物清除。硫氧还蛋白还原酶在皮肤匀浆的氮氧化物还原中没有显著参与。在小鼠表皮表面，一种在哺乳动物硫氧还蛋白还原酶存在下稳定的阳离子二氢吡咯氧基氮氧化物很容易被还原。表皮还原受到锌、N-乙基马来酰亚胺和加热（70℃，5分钟）的抑制。至少对于小鼠表皮来说，多种氮氧化物的还原是一个复杂的现象，涉及酶促和非酶促机制，不能用作例如硫氧还蛋白还原酶等酶的特异性测定方法。该研究表明表皮含有一种有效的抗氧化系统，该系统能清除对抗坏血酸敏感的哌啶氧基氮氧化物以及以二氢吡咯氧基氮氧化物为代表的更多还原性自由基。
