基于微阵列聚焦电泳的糖尿病血样临床阳离子交换高效液相色谱图中血红蛋白A_(3)峰位置推测  

Speculation of hemoglobin A_(3) peak position in clinical cation exchange high performance liquid chromatogram of the diabetic blood sample with microarray isoelectric focusing

作　　者：郭泽华 罗芳 李思 樊柳荫[3] 伍贻新[4] 曹成喜 GUO Zehua;LUO Fang;LI Si;FAN Liuyin;WU Yixin;CAO Chengxi(Department of Instrument Science and Engineering, School of Electronic Information and Electrical Engineering, Shanghai Jiao Tong University, Shanghai 200240, China;State Key Laboratory of Microbial Metabolism, School of Life Science and Biotechnology, Shanghai Jiao Tong University, Shanghai 200240, China;Student Innovation Center, Shanghai Jiao Tong University, Shanghai 200240, China;Endocrine Institute, Ruijin Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai 200230, China)

机构地区：[1]上海交通大学电子信息与电气工程学院仪器科学与工程系,上海200240 [2]上海交通大学生命科学技术学院,微生物代谢国家重点实验室,上海200240 [3]上海交通大学学生创新中心,上海200240 [4]上海交通大学医学院附属瑞金医院内分泌科,上海200230

出　　处：《色谱》2021年第11期1273-1278,共6页Chinese Journal of Chromatography

基　　金：国家自然科学基金(31727801,22074091);国家科学仪器设备重大开发项目(2011YQ030139).

摘　　要：血红蛋白A_(1c)(HbA_(1c))是糖尿病诊断的关键生物标志物,目前其常用的分析方法为阳离子交换高效液相色谱法(CX-HPLC,5/50 mm分离柱),此方法虽然具有稳定、快捷与自动化等众多优点,但临床CX-HPLC(VARIANTⅡsystem)谱图中仍存在未知峰,尤其干扰HbA1c准确测定的谷胱甘肽化血红蛋白A_(3)(HbA_(3))在色谱图中的相对位置仍不清楚。针对这一问题,该文以人新鲜血液为样本,首先利用低分辨CX-HPLC对血样进行分析,提示未知峰P3存在。然后通过微阵列等电聚焦(IEF)电泳和高分辨阳离子交换HPLC(Mono-S 5/50 mm分离柱)对血样的主要血红蛋白(Hb)成分进行分析,提示未知峰P3为HbA3峰。随后,通过血红蛋白谷胱甘肽化实验,利用HbA_(1c)峰降低、HbA_(3)峰明显增强这一信息,进一步推测未知峰P3即是HbA_(3)峰,其在CX-HPLC中的保留时间在1.50 min左右。最后,结合前期研究讨论了体内应激情况下Hb谷胱甘肽化对HbA_(1c)检测的影响。该研究丰富了CX-HPLC的未知峰P3的色谱信息,为更精准诊断糖尿病提供了有价值的参考。Hemoglobin A_(1c)(HbA_(1c))is a major component of glycated hemoglobin in human red blood cells.It has been proven to be a significant biomarker for the diagnosis of diabetes;its content in fresh red cells in diabetes blood reflects the average level of blood glucose over the previous three months.Thus,HbA1c level has been used for the assessment of long-term glycemic control in diabetes;the level of 6.5%HbA_(1c) has been certified as a critical cut-off for the diabetes diagnosis.The current commonly used method for HbA_(1c) quantification is based on cation-exchange high performance liquid chromatography(CX-HPLC).The method has advantages such as high stability,rapidity,and automation,but there are still some unidentified peaks of Hb species in CX-HPLC(VARIANTⅡsystem);in particular,the presence of HbA_(3)(a glutathiolated Hb)affects the accurate determination of HbA1c.HbA_(3) is usually present in healthy adult blood samples at 2%-4%,but the concentration of HbA_(3) increases due to the protection of erythrocytes from oxidation,resulting in decreased HbA1c.However,the relative location of the HbA_(3) peak in the CX-HPLC clinical chromatogram has not been established.To address this issue, we extracted Hb species from fresh blood samples obtained from a hospital in an anaerobic environment to avoid possible redox reactions of Hb and glutathione. After the extraction, the Hb samples were analyzed using two methods: a low-resolution CX-HPLC(5/50 mm column) currently used for diabetes diagnosis and a high-resolution cationic exchange HPLC(Mono-S 5/50 mm column), to identify the peak corresponding to HbA3. The CX-HPLC analysis of fresh blood samples indicated that the unknown peak P3 located between HbA1 c and HbA0 peaks corresponded to the HbA3 peak between HbA1 c and HbA0 in the Mono-S-HPLC. Microarray isoelectric focusing(IEF) was used for the micro-preparation of HbA3, HbA1 c, and HbA0 in healthy blood samples;then, the micro-prepared species of HbA3, HbA1 c, and HbA0 were individually identified via Mono-S-HPL

关 键 词：阳离子交换高效液相色谱 等电聚焦 血红蛋白A_(3) 血红蛋白A_(1c) 血样 糖尿病 

分 类 号：O658[理学—分析化学]