Nagai Ryoji, Deemer Elizabeth K, Brock Jonathan W, Thorpe Suzanne R, Baynes John W
Department of Chemistry and Biochemistry, Graduate Science Research Center, University of South Carolina, Columbia, SC 29208, USA.
Ann N Y Acad Sci. 2005 Jun;1043:146-50. doi: 10.1196/annals.1333.018.
Posttranslational modifications, such as advanced glycoxidation and lipoxidation end products (AGE/ALEs), are implicated in the pathogenesis of diabetic complications and atherosclerosis. Recent studies have demonstrated that AGE/ALEs are generated not only in extracellular matrix proteins, but also in intracellular proteins from metabolic intermediates. In this study we investigate the effect of glucose concentration on the formation of the AGE/ALEs, Nepsilon-(carboxymethyl)lysine (CML), Nepsilon-(carboxyethyl)lysine (CEL), S-(carboxymethyl)cysteine (CMC), and S-(2-succinyl)cysteine (2SC) in erythrocytes as a function of glucose concentration. Human erythrocytes (10% hematocrit) were incubated in Dulbecco's modified Eagle's medium (DMEM) containing 5 mM or 30 mM glucose for 5 days at 37 degrees C. Globin was recovered by precipitation with 0.25 M HCl in acetone. Following acid hydrolysis, amino acids were converted to their trifluoroacetyl methyl ester derivatives and analyzed by GC/MS/MS. The CML and CEL content of globin increased in a time- and glucose-dependent manner and also increased 1.3- and 1.8-fold, respectively, in incubations containing 30 mM glucose; whereas CMC and 2SC content did not change during the five-day incubations. Furthermore, CEL content of globin in erythrocytes incubated with 30 mM was the highest in the other AGEs, indicating that methylglyoxal may play a major role in AGE formation in erythrocytes. The erythrocyte system should be useful for cellular screening of the efficacy of inhibitors of AGE/ALE formation.
翻译后修饰，如晚期糖基化终末产物和晚期脂质氧化终末产物（AGE/ALEs），与糖尿病并发症和动脉粥样硬化的发病机制有关。最近的研究表明，AGE/ALEs不仅在细胞外基质蛋白中产生，也在细胞内由代谢中间体产生。在本研究中，我们研究了葡萄糖浓度对红细胞中AGE/ALEs、Nε-（羧甲基）赖氨酸（CML）、Nε-（羧乙基）赖氨酸（CEL）、S-（羧甲基）半胱氨酸（CMC）和S-（2-琥珀酰）半胱氨酸（2SC）形成的影响，该影响是葡萄糖浓度的函数。将人红细胞（血细胞比容为10%）在含有5 mM或30 mM葡萄糖的杜氏改良 Eagle 培养基（DMEM）中于37℃孵育5天。通过用0.25 M HCl在丙酮中沉淀回收球蛋白。酸水解后，氨基酸转化为其三氟乙酰甲酯衍生物，并通过气相色谱/串联质谱（GC/MS/MS）进行分析。球蛋白的CML和CEL含量呈时间和葡萄糖依赖性增加，在含有30 mM葡萄糖的孵育中分别增加了1.3倍和1.8倍；而CMC和2SC含量在为期五天的孵育过程中没有变化。此外，在30 mM葡萄糖孵育的红细胞中，球蛋白的CEL含量在其他AGEs中最高，这表明甲基乙二醛可能在红细胞中AGE形成中起主要作用。红细胞系统应有助于对AGE/ALE形成抑制剂的疗效进行细胞筛选。
