Kim So Ra, Jockusch Steffen, Itagaki Yasuhiro, Turro Nicholas J, Sparrow Janet R
Department of Ophthalmology, Columbia University, 630 W.168th Street, NY 10032, USA.
Exp Eye Res. 2008 Jun;86(6):975-82. doi: 10.1016/j.exer.2008.03.016. Epub 2008 Mar 28.
A2E is one of the bis-retinoid pyridinium compounds that accumulate as lipofuscin pigments in retinal pigment epithelial (RPE) cells in association with aging and in some inherited forms of retinal degeneration. Here we observed that 430nm irradiation of A2E in the presence of the spin trap DMPO, led to the appearance of a superoxide dismutase-inhibitable electron paramagnetic resonance (EPR) spectrum characteristic of DMPO-OH; this finding was indicative of hydroxyl radical (OH) formation following initial spin trapping of superoxide anion by DMPO. We also observed an increase in dihydroethidium (HEt) fluorescence and luminol-based chemiluminescence that on the basis of inhibition by superoxide dismutase, was indicative of superoxide anion generation when A2E was irradiated at 430nm in cell-free systems. Nevertheless, while A2E was readily oxidized in the presence of a singlet oxygen generator, superoxide anion did not serve to oxidize A2E. Specifically, by HPLC quantitation and FAB-mass spectroscopy, there was no evidence of A2E oxidation when A2E was incubated with a superoxide anion generator (xanthine/xanthine oxidase) in a variety of solvents (100% PBS, 30% DMSO in PBS, 100% MeOH and CHCl3) or in the presence of detergent. On the other hand, however, peroxy-A2E, an oxidized form of A2E with an endoperoxide moiety on the short-arm of the molecule, readily underwent further oxygen addition when incubated with xanthine/xanthine oxidase. Superoxide anion may be generated by irradiation of A2E but is not involved in the early events that oxidize A2E. Superoxide can contribute to the further oxidation of already-oxidized A2E.
A2E是双视黄醛吡啶鎓化合物之一，随着年龄增长以及在某些遗传性视网膜变性中，它会作为脂褐素色素在视网膜色素上皮（RPE）细胞中积累。在此我们观察到，在自旋捕获剂DMPO存在的情况下，用430nm光照射A2E，会产生一种超氧化物歧化酶可抑制的电子顺磁共振（EPR）光谱，该光谱具有DMPO-OH的特征；这一发现表明，超氧阴离子首先被DMPO自旋捕获后会形成羟基自由基（OH）。我们还观察到二氢乙锭（HEt）荧光和基于鲁米诺的化学发光增加，基于超氧化物歧化酶的抑制作用，这表明在无细胞体系中用430nm光照射A2E时会产生超氧阴离子。然而，虽然在单线态氧发生器存在的情况下A2E很容易被氧化，但超氧阴离子并不会氧化A2E。具体而言，通过高效液相色谱定量和快原子轰击质谱分析，当A2E在多种溶剂（100%磷酸盐缓冲盐水、磷酸盐缓冲盐水中30%二甲基亚砜、100%甲醇和氯仿）中或在去污剂存在的情况下与超氧阴离子发生器（黄嘌呤/黄嘌呤氧化酶）孵育时，没有证据表明A2E被氧化。另一方面，然而，过氧-A2E是A2E的一种氧化形式，其分子短臂上有一个内过氧化物部分，当与黄嘌呤/黄嘌呤氧化酶孵育时很容易进一步被氧化。超氧阴离子可能由A2E的照射产生，但不参与氧化A2E的早期事件。超氧化物可以促进已氧化的A2E的进一步氧化。
