Detection of noncarboxymethyllysine and carboxymethyllysine advanced glycation end products (AGE) in serum of diabetic patients
Detection of noncarboxymethyllysine and carboxymethyllysine advanced glycation end products (AGE) in serum of diabetic patients
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DOI：
10.1007/bf03402128
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发表时间：
1999-06-01
影响因子：
5.7
通讯作者：
Koike, T
中科院分区：
文献类型：
--
作者：
Takeuchi, M;Makita, Z;Koike, T
背景资料：美拉德反应的晚期阶段导致晚期糖基化终产物（AGE）的形成，在糖尿病患者的血管病发病机制和衰老过程中起重要作用。N-β-（羧甲基）赖氨酸（CML）被认为是许多目前可用的AGE抗体的重要表位。然而，最近的研究结果表明，CML的主要来源可能是通过糖基化以外的途径。CML和非CML AGE之间的区别可能会增加我们对体内AGE形成的理解。在本研究中，我们制备了针对CML和非CML AGE.Materials和方法的抗体：AGE-兔血清白蛋白与葡萄糖孵育4，8，12周制备的用于免疫家兔，并获得高滴度的AGE特异性抗血清，没有对载体蛋白的亲和力。为了分离CML和非CML AGE抗体，将抗AGE抗血清在与AGE-BSA和CML-BSA偶联的柱上进行亲和层析。获得了两种不同的抗体，一种与CML特异性反应，另一种与非CML AGE反应。用竞争ELISA法测定了66例非尿毒症2型糖尿病患者的慢性粒细胞白血病（CML）和非CML晚期糖基化终产物（AGE）水平。在接受血液透析的糖尿病患者的血清中，评估了非CML AGE和CML检测的血液透析的粒径分布和清除率。结果如下：2型糖尿病患者血清非CML、AGE水平与2个月内平均空腹血糖水平显著相关（r = 0.498，p <0.0001）或前1个月与糖化血红蛋白（HbA1c）呈显著正相关（r = 0.375，p = 0.0019），但CMI、AGE水平与这些临床指标无相关性。CML和非CML AGE检测为4个峰，表观分子量分别为200、65、1.15和0.85 kD。血液透析治疗不影响高分子量蛋白组分。尽管低分子量肽级分结果表明，非CML AGE-和CML-肽组分在透析前后（280 nm处的吸光度和荧光）均降低，但无差异（1.15和0.85 kD组分）。我们认为CML和非CML AGE都存在于血液中，非CML AGE也存在于血液中。在研究年龄相关疾病的病理生理学时，应更密切地评价CML AGE而不是CML AGE。
Background: The advanced stage of the Maillard reaction, which leads to the formation of advanced glycation end products (AGE), plays an important role in the pathogenesis of angiopathy in diabetic patients and in the aging process. N-epsilon-(carboxymethyl)lysine (CML) is thought to be an important epitope for many of currently available AGE antibodies. However, recent findings have indicated that a major source of CML may be by pathways other than glycation. A distinction between CML and non-CML AGE may increase our understanding of AGE formation in vivo. In the present study, we prepared antibodies directed against CML and non-CML AGE.Materials and Methods: AGE-rabbit serum albumin prepared by 4, 8, and 12 weeks of incubation with glucose was used to immunize rabbits, and a high-titer AGE-specific antiserum was obtained without affinity for the carrier protein. To separate CML and non-CML AGE antibodies, the anti-AGE antiserum was subjected to affinity chromatography on a column coupled with AGE-BSA and CML-BSA. Two different antibodies were obtained, one reacting specifically with CML and the other reacting with non-CML AGE. Circulating levels of CML and non-CML AGE were measured in 66 type 2 diabetic patients without uremia by means of the competitive ELISA. Size distribution and clearance by hemodialysis detected by non-CML AGE and CML were assessed in serum from diabetic patients on hemodialysis. Results: The serum non-CML, AGE level in type 2 diabetic patients was significantly correlated with the mean fasting blood glucose level over the previous 2 months (r = 0.498, p < 0.0001) or the previous 1 month (r = 0.446, p = 0.0002) and with HbA(1c) (r = 0.375, p = 0.0019), but the CMI, AGE level was not correlated with these clinical parameters. The CML and non-CML AGE were detected as four peaks with apparent molecular weights of 200, 65, 1.15, and 0.85 kD. The hemodialysis treatment did not affect the high-molecular-weight protein fractions. Although the low-molecular-weight peptide fractions (absorbance at 280 nm and fluorescence) were decreased by hemodialysis, there was no difference before and after dialysis in the non-CML AGE- and CML-peptide fractions (1.15 and 0.85 kD fractions).Conclusions: We propose that both CML and non-CML AGE are present in the blood and that non-CML AGE rather than CML AGE should be more closely evaluated when investigating the pathophysiology of AGE-related diseases.