Price D L, Rhett P M, Thorpe S R, Baynes J W
Department of Chemistry and Biochemistry, University of South Carolina, Columbia, South Carolina 29208, USA.
J Biol Chem. 2001 Dec 28;276(52):48967-72. doi: 10.1074/jbc.M108196200. Epub 2001 Oct 24.
The advanced glycation end-product (AGE) hypothesis proposes that accelerated chemical modification of proteins by glucose during hyperglycemia contributes to the pathogenesis of diabetic complications. The two most commonly measured AGEs, N(epsilon)-(carboxymethyl)lysine and pentosidine, are glycoxidation products, formed from glucose by sequential glycation and autoxidation reactions. Although several compounds have been developed as AGE inhibitors and are being tested in animal models of diabetes and in clinical trials, the mechanism of action of these inhibitors is poorly understood. In general, they are thought to function as nucleophilic traps for reactive carbonyl intermediates in the formation of AGEs; however alternative mechanisms of actions, such as chelation, have not been rigorously examined. To distinguish between the carbonyl trapping and antioxidant activity of AGE inhibitors, we have measured the chelating activity of the inhibitors by determining the concentration required for 50% inhibition of the rate of copper-catalyzed autoxidation of ascorbic acid in phosphate buffer. All AGE inhibitors studied were chelators of copper, as measured by inhibition of metal-catalyzed autoxidation of ascorbate. Apparent binding constants for copper ranged from approximately 2 mm for aminoguanidine and pyridoxamine, to 10-100 microm for carnosine, phenazinediamine, OPB-9195 and tenilsetam. The AGE-breakers, phenacylthiazolium and phenacyldimethylthiazolium bromide, and their hydrolysis products, were among the most potent inhibitors of ascorbate oxidation. We conclude that, at millimolar concentrations of AGE inhibitors used in many in vitro studies, inhibition of AGE formation results primarily from the chelating or antioxidant activity of the AGE inhibitors, rather than their carbonyl trapping activity. Further, at therapeutic concentrations, the chelating activity of AGE inhibitors and AGE-breakers may contribute to their inhibition of AGE formation and protection against development of diabetic complications.
晚期糖基化终产物（AGE）假说提出，在高血糖期间葡萄糖对蛋白质的加速化学修饰促成了糖尿病并发症的发病机制。两种最常检测的AGEs，N（ε）-（羧甲基）赖氨酸和戊糖苷，是糖氧化产物，由葡萄糖通过连续的糖基化和自氧化反应形成。尽管已经开发了几种化合物作为AGE抑制剂，并正在糖尿病动物模型和临床试验中进行测试，但这些抑制剂的作用机制仍知之甚少。一般来说，它们被认为是在AGEs形成过程中作为亲核陷阱捕获活性羰基中间体；然而，其他作用机制，如螯合作用，尚未得到严格研究。为了区分AGE抑制剂的羰基捕获和抗氧化活性，我们通过测定在磷酸盐缓冲液中50%抑制抗坏血酸铜催化自氧化速率所需的浓度来测量抑制剂的螯合活性。通过抑制抗坏血酸的金属催化自氧化来测量，所有研究的AGE抑制剂都是铜的螯合剂。铜的表观结合常数范围从氨基胍和吡哆胺的约2 mM到肌肽、吩嗪二胺、OPB-9195和替尼西坦的10 - 100 μM。AGE裂解剂苯甲酰噻唑鎓和苯甲酰二甲基噻唑鎓溴化物及其水解产物是抗坏血酸氧化的最有效抑制剂之一。我们得出结论，在许多体外研究中使用的毫摩尔浓度的AGE抑制剂下，AGE形成的抑制主要源于AGE抑制剂的螯合或抗氧化活性，而不是它们的羰基捕获活性。此外，在治疗浓度下，AGE抑制剂和AGE裂解剂的螯合活性可能有助于它们抑制AGE形成并预防糖尿病并发症的发展。
