色谱

色谱 ›› 2021, Vol. 39 ›› Issue (11): 1273-1278.DOI: 10.3724/SP.J.1123.2020.12033

• 技术与应用 • 上一篇    

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A3峰位置推测

郭泽华1, 罗芳2, 李思2, 樊柳荫3,*(), 伍贻新4, 曹成喜1,*()   

  1. 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
  • 收稿日期:2020-12-31 出版日期:2021-11-08 发布日期:2021-05-12
  • 通讯作者: 樊柳荫,曹成喜
  • 作者简介:E-mail: cxcao@sjtu.edu.cn(曹成喜).
    * E-mail: lyfan@sjtu.edu.cn(樊柳荫);
  • 基金资助:
    国家自然科学基金(31727801);国家自然科学基金(22074091);国家科学仪器设备重大开发项目(2011YQ030139)

Speculation of hemoglobin A3 peak position in clinical cation exchange high performance liquid chromatogram of the diabetic blood sample with microarray isoelectric focusing

GUO Zehua1, LUO Fang2, LI Si2, FAN Liuyin3,*(), WU Yixin4, CAO Chengxi1,*()   

  1. 1. Department of Instrument Science and Engineering, School of Electronic Information and Electrical Engineering, Shanghai Jiao Tong University, Shanghai 200240, China
    2. State Key Laboratory of Microbial Metabolism, School of Life Science and Biotechnology, Shanghai Jiao Tong University, Shanghai 200240, China
    3. Student Innovation Center, Shanghai Jiao Tong University, Shanghai 200240, China
    4. Endocrine Institute, Ruijin Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai 200230, China
  • Received:2020-12-31 Online:2021-11-08 Published:2021-05-12
  • Contact: FAN Liuyin,CAO Chengxi
  • Supported by:
    National Natural Science Foundation of China(31727801);National Natural Science Foundation of China(22074091);National Scientific Instruments Key Development Project(2011YQ030139)

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摘要：

血红蛋白A1c(HbA1c)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(VARIANT Ⅱ system)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A3(HbA3)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HbA1c峰降低、HbA3峰明显增强这一信息,进一步推测未知峰P3即是HbA3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HbA1c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。

关键词: 阳离子交换高效液相色谱, 等电聚焦, 血红蛋白A3, 血红蛋白A1c, 血样, 糖尿病

Abstract:

Hemoglobin A1c (HbA1c) 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% HbA1c has been certified as a critical cut-off for the diabetes diagnosis. The current commonly used method for HbA1c 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 (VARIANT Ⅱ system); in particular, the presence of HbA3 (a glutathiolated Hb) affects the accurate determination of HbA1c. HbA3 is usually present in healthy adult blood samples at 2%-4%, but the concentration of HbA3 increases due to the protection of erythrocytes from oxidation, resulting in decreased HbA1c. However, the relative location of the HbA3 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 HbA1c and HbA0 peaks corresponded to the HbA3 peak between HbA1c and HbA0 in the Mono-S-HPLC. Microarray isoelectric focusing (IEF) was used for the micro-preparation of HbA3, HbA1c, and HbA0 in healthy blood samples; then, the micro-prepared species of HbA3, HbA1c, and HbA0 were individually identified via Mono-S-HPLC. The results of the CX-HPLC, Mono-S-HPLC, and microarray IEF experiments indicated that the P3 peak might correspond to HbA3. To confirm this, glutathiolated Hb samples were synthesized via acetylphenylhydrazine and analyzed using both the Mono-S- and CX-HPLC systems. The results showed that the content of both glutaminated hemoglobin of HbA3 in Mono-S-HPLC and P3 in CX-HPLC increased, implying the peak of P3 with the retention time of 1.50 min in CX-HPLC was the peak corresponding to HbA3 in Mono-S-HPLC and microarray IEF.
Based on the above experiments and our previous results, the influence of HbA3 on both the analysis of HbA1c in blood samples and the diabetes diagnosis needs to be considered and discussed. The study results are significant for the tentative assignment of peak P3 and for offering more information on diabetes diagnosis using CX-HPLC in the clinical setting.

Key words: cation exchange high performance liquid chromatography (CX-HPLC), isoelectric focusing (IEF), hemoglobin A3 (HbA3), hemoglobin A1c (HbA1c), blood sample, diabetes

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