Saxena P, Saxena A K, Monnier V M
Institute of Pathology, Case Western Reserve University, Cleveland, OH 44106, USA.
Exp Eye Res. 1996 Nov;63(5):535-45. doi: 10.1006/exer.1996.0144.
In contrast to conventional view that glucose is the sole glycating agent, ascorbate has now emerged as a potential precursor of advanced glycation products in lenses during cataractogenesis, owing to the high concentration present in human lens. The effects of high hexose environment in vitro and in vivo on the disruption of redox equilibrium of ascorbate (ASA) to dehydroascorbate (DHA), which is required for ascorbate-mediated crystallin modification by the Maillard reaction during cataractogenesis were examined. Organ culture experiments were performed with rat lenses that were first exposed to high galactose levels in vitro and in vivo and then incubated with 1-14C-labeled ASA, DHA or DKG (2,3-diketogulonic acid). Formation of ASA degradation products as a function of time was assessed by radiometric TLC method. Upon incubation with ASA or DHA, an elevated level of the degradation product, DKG, was detected in lenses exposed to galactose in vivo and in vitro. ASA uptake was significantly enhanced in the galactosemic lenses as compared to controls (P = 0.01). Regeneration of ASA from DHA in both galactose treated and galactosemic lenses was impaired when compared to control lens which completely converted DHA from the medium into ASA. Surprisingly, the galactose exposed lenses showed enhanced permeability to DKG which was picked up readily from the medium in contrast to normal healthy lenses which remained impermeable to DKG. Galactose exposed lenses both in vitro and in vivo showed a 5-9-fold increase in crystallin bound Schiff base-linked radioactivity when incubated with 1-14C-labeled ASA or DHA. As a preamble to the question of whether lens pigmentation predisposes towards ascorbate oxidation, lens homogenate from normal young and old pigmented cataractous lenses were incubated with [1-14C]ASA. After 2 days, ASA levels were found to have decreased by 74% and DKG levels increased by 48% in brunescent lens as compared to the young lens. These data demonstrated that profound abnormalities in ASA metabolism exist in lenses exposed to a high sugar environment suggestive of a breakdown of the redox equilibrium of ASA to DHA and a loss of membrane permeability barrier for DKG. The latter would further contribute toward a ASA-catalysed Maillard reaction in the redox impaired lens.
与葡萄糖是唯一糖化剂的传统观点不同，由于人晶状体中抗坏血酸的高浓度存在，它现已成为白内障形成过程中晶状体晚期糖基化终产物的潜在前体。研究了体外和体内高己糖环境对白内障形成过程中抗坏血酸（ASA）向脱氢抗坏血酸（DHA）氧化还原平衡破坏的影响，这种平衡是抗坏血酸通过美拉德反应介导晶状体蛋白修饰所必需的。用大鼠晶状体进行器官培养实验，这些晶状体先在体外和体内暴露于高半乳糖水平，然后与1-14C标记的ASA、DHA或DKG（2,3-二酮古洛糖酸）一起孵育。通过放射性薄层色谱法评估ASA降解产物随时间的形成情况。与ASA或DHA一起孵育后，在体内和体外暴露于半乳糖的晶状体中检测到降解产物DKG的水平升高。与对照组相比，半乳糖血症晶状体中的ASA摄取显著增强（P = 0.01）。与完全将培养基中的DHA转化为ASA的对照晶状体相比，半乳糖处理的晶状体和半乳糖血症晶状体中DHA再生为ASA的过程均受到损害。令人惊讶的是，与对DKG保持不透性 的正常健康晶状体相比，暴露于半乳糖的晶状体对DKG的通透性增强，DKG很容易从培养基中摄取。体外和体内暴露于半乳糖的晶状体在与1-14C标记的ASA或DHA孵育时，晶状体蛋白结合的席夫碱连接放射性增加了5-9倍。作为晶状体色素沉着是否易导致抗坏血酸氧化问题的前言，将正常年轻和老年色素性白内障晶状体的匀浆与[1-14C]ASA一起孵育。2天后，与年轻晶状体相比，褐色晶状体中的ASA水平下降了74%，DKG水平增加了48%。这些数据表明，暴露于高糖环境的晶状体中存在抗坏血酸代谢的严重异常，提示ASA向DHA的氧化还原平衡破坏以及对DKG的膜通透性屏障丧失。后者将进一步促进氧化还原受损晶状体中ASA催化的美拉德反应。
