Detemination of The Main Advanced Glycation End Product of The Maillard Reaction and Its Significance as A Biochemical Marker for Diabetic Complications

葡萄糖与蛋白质在体外孵育可形成早期产物，如希夫碱和阿玛多里产物。在进一步孵育后，这些早期产物导致形成晚期糖基化终产物（AGE），其特征是荧光，棕色和分子内和分子间交联。最近使用抗age抗体的免疫学研究表明，age修饰在衰老和年龄增强的疾病过程（如动脉粥样硬化和糖尿病并发症）中具有潜在作用。为了解决这一问题，需要确定体内表达的主要age结构。本研究从age -赖氨酸衍生物中分离出age -主要结构并测定其化学结构。以下是通过两年的项目得到的结果age样品经α - toyl -lysine-methyl ester与葡萄糖长期孵育后得到，并从中纯化出主要荧光化合物X1。化学分析通过质谱和1H-NMR和13C-NMR成功鉴定其结构为1,7-二取代-5-（1,2,3,4-四羟基丁基）-1,4-二氢-4-氧-1,7-萘基，表明两个赖氨酸残基是由两个葡萄糖分子衍生的吡啶结构交联的使用兔多克隆抗X1抗体的免疫学研究表明，X1存在于(i)人类晶状体蛋白，（ii）泡沫细胞以及人类动脉粥样硬化病变的细胞外间隙，（iii）糖尿病肾病的肾小球和肾小管，（iv）透析相关淀粉样变性的腕管沉积物（β 2微球蛋白），(v)光化弹性变性的皮肤真皮弹性蛋白纤维中。这些结果表明，X1是体内主要的age结构之一，在衰老和/或年龄增强的疾病过程中起重要作用，如糖尿病并发症和血管壁动脉粥样硬化。

Incubation of glucose with proteins in vitro results in formation of early-products such as Schiff base and Amadori product. Upon further incubations, these early-products leads to formation of advanced glycation end products (AGE) which are characterized by fluorescence, brown color and intra-and intermolecular crosslinking. Recent immunological studies using anti-AGE antibody suggest a potential role of AGE-modification in aging and age-enhanced disease processes such as atherosclerosis and diabetic complications. To solve this suggestion, a main AGE-structure (s) expressed in vivo is to be determined. In the present study, we isolated the main AGE-structure from AGE-lysine derivative and determined its chemical structure.The following results were obtained by two-years projects.[1] The AGE-sample was obtained after long-term incubation of alpha-tosyl-lysine-methyl ester with glucose and the main fluorescent compound named as X1 was purified from this sample. Chemical analyzes by mass spectroscopy and 1H-NMR and 13C-NMR successfully identified its structure as 1,7-disubstituted-5- (1,2,3,4-tetrahydroxybutyl) -1,4-dihydro-4-oxo-1,7-naphthyridinium cation, indicating two lysine residues are crosslinked by pyridinium structure derived from two glucose molecules.[2] Immunological studies using the rabbit polyclonal anti-X1 antibody demonstrated the presence of X1 in (i) human lens proteins, (ii) foam cells as well as in extracellular spaces in human atherosclerotic lesions, (iii) glomerulus and renal tubules of diabetic nephropathy, (iv) in Carpal tunnel deposits (beta2-microglobulin) of dialysis-related amyloidosis, (v) elastin fibers of skin dermis in actinic elastosis. These results indicate that X1, one of the main AGE-structures in vivo, plays an important role in aging and/or age-enhanced disease processes such as diabetic complications and atherosclerosis of vascular walls.