Saxena P, Saxena A K, Cui X L, Obrenovich M, Gudipaty K, Monnier V M
Institute of Pathology, Case Western Reserve University, School of Medicine, Cleveland, Ohio 44106, USA.
Invest Ophthalmol Vis Sci. 2000 May;41(6):1473-81.
With age, human lens crystallins become more pigmented, oxidized, modified by ascorbate oxidation and advanced glycation end products (AGEs), and bind copper. The hypothesis was tested that the major AGE and ascorbylation product in the human lens, N(epsilon)-carboxymethyl-L-lysine (CML), has an EDTA-like structure, which may predispose it to bind redox active copper.
Young, old, and cataractous human lens protein fractions were glycated with ascorbic acid and tested for their ability to bind Cu(II) by atomic absorption spectroscopy and oxidize (14C1)-ascorbate by radiometric thin-layer chromatography method. AGEs were assayed by high-performance liquid chromatography (HPLC). CML-rich proteins were immunoprecipitated from young, old, and cataractous crystallins using affinity-purified CML antibody and tested for their ability to oxidize ascorbate and generate hydroxyl radicals in the presence of H2O2 using 5,5'-dimethyl-1-pyrroline-N-oxide (DMPO) spin-trap and EPR spectroscopy.
Ascorbate oxidizing activity at 24 hours of native crystallins was significantly increased in both the water soluble (WS; P < 0.001) and insoluble (WIS; P < 0.05) fractions from cataractous and normal lenses. The chelator DTPA completely prevented oxidation up to 24 hours of incubation but less effectively thereafter. Mean endogenous Cu content in pooled young, old, and cataract fractions increased from 0.016 to 0.026 nmol/mg protein, respectively, in WS (P < 0.05) and WIS (P < 0.001) fractions, and Cu(II) binding was 20% to 30% increased in cataractous versus old and young lenses in WS (P < 0.01) and WIS (P < 0.001) fractions. Mean levels of the AGEs, CML, and pentosidine were markedly elevated in WS and WIS fractions from cataractous versus old or young crystallins (20% to severalfold, P < 0.05 to P < 0.001). In a separate experiment, protein-bound Fe was not elevated. Crystallins ascorbylated in vitro showed an increase in CML as well as Cu(II) binding. CML-rich proteins (immunoprecipitated from cataractous lenses) oxidized ascorbate approximately 4 times faster than similar proteins from young and old normal lenses (P < 0.01) and generated hydroxyl radicals in the presence of H2O2 and DMPO.
The association between CML formation, copper binding, and generation of free radicals by cataractous lens crystallins can be duplicated by ascorbylation in vitro. These effects are only in part attributable to CML itself, and other modifications (AGEs, conformational changes) may participate in the process. A vicious cycle between AGE formation, lipoxidation, and metal binding may exist in the aging lens, suggesting that chelation therapy could be beneficial in delaying cataractogenesis.
随着年龄增长，人晶状体晶状体蛋白色素沉着增加、被氧化、被抗坏血酸氧化及晚期糖基化终产物（AGEs）修饰，并与铜结合。本研究检验了如下假说：人晶状体中主要的AGE和抗坏血酸化产物N(ε)-羧甲基-L-赖氨酸（CML）具有类似乙二胺四乙酸（EDTA）的结构，这可能使其易于结合具有氧化还原活性的铜。
用抗坏血酸使年轻、年老及白内障患者的晶状体蛋白组分发生糖基化，并通过原子吸收光谱法检测其结合Cu(II)的能力，通过放射性薄层色谱法检测其氧化(14C1)-抗坏血酸的能力。采用高效液相色谱法（HPLC）检测AGEs。使用亲和纯化的CML抗体从年轻、年老及白内障晶状体蛋白中免疫沉淀富含CML的蛋白，并使用5,5'-二甲基-1-吡咯啉-N-氧化物（DMPO）自旋捕集和电子顺磁共振波谱法检测其在H2O2存在下氧化抗坏血酸并产生羟自由基的能力。
白内障和正常晶状体的水溶性（WS；P < 0.001）和不溶性（WIS；P < 0.05）组分中，天然晶状体蛋白在24小时时的抗坏血酸氧化活性均显著增加。螯合剂二乙烯三胺五乙酸（DTPA）在孵育24小时内完全抑制了氧化，但之后效果较差。在WS（P < 0.05）和WIS（P < 0.001）组分中，年轻、年老及白内障混合组分的平均内源性铜含量分别从0.016 nmol/mg蛋白增加至0.026 nmol/mg蛋白，与年老和年轻晶状体相比，白内障晶状体在WS（P < 0.01）和WIS（P < 0.001）组分中的Cu(II)结合增加了20%至30%。与年老或年轻晶状体蛋白的WS和WIS组分相比，白内障晶状体蛋白的AGEs、CML和戊糖苷平均水平显著升高（20%至数倍，P < 0.05至P < 0.001）。在另一项实验中，蛋白结合铁未升高。体外抗坏血酸化的晶状体蛋白显示CML以及Cu(II)结合增加。从白内障晶状体中免疫沉淀的富含CML的蛋白氧化抗坏血酸的速度比来自年轻和年老正常晶状体的类似蛋白快约4倍（P < 0.01），并在H2O2和DMPO存在下产生羟自由基。
白内障晶状体蛋白中CML形成、铜结合及自由基产生之间的关联可通过体外抗坏血酸化复制。这些效应仅部分归因于CML本身，其他修饰（AGEs、构象变化）可能参与该过程。老化晶状体中可能存在AGE形成、脂氧化和金属结合之间的恶性循环，提示螯合疗法可能有助于延缓白内障的发生。
