Xu Xiaohui, Li Daoyuan, Chi Lequan, Du Xuzhao, Bai Xue, Chi Lianli
National Glycoengineering Research Center, Shandong University, Jinan 250100, China; State Key Laboratory of Microbial Technology, Shandong University, Jinan 250100, China.
School of Marine and Atmospheric Sciences, Stony Brook University, Stony Brook, NY 11794, USA.
Carbohydr Res. 2015 Apr 30;407:26-33. doi: 10.1016/j.carres.2015.01.016. Epub 2015 Feb 2.
Low molecular weight heparins (LMWHs) are linear and highly charged carbohydrate polymers prepared by chemical or enzymatic depolymerization of heparin. Compared to unfractionated heparin (UFH), LMWHs are prevalently used as clinical anticoagulant drugs due to their lower side effects and better bioavailability. The work presented herein provides a rapid and powerful fragment mapping method for structural characterization of LMWHs. The chain fragments of two types of LMWHs, enoxaparin and nadroparin, were generated by controlled enzymatic digestion with each of heparinase I (Hep I, Enzyme Commission (EC) # 4.2.2.7), heparinase II (Hep II, no EC # assigned) and heparinase III (Hep III, EC # 4.2.2.8). Reversed phase ion pair high performance liquid chromatography (RPIP-HPLC) coupled with electrospray ion trap time-of-flight mass spectrometry (ESI-IT-TOF-MS) was used to profile the oligosaccharide chains ranging from disaccharides to decasaccharides. A database containing all theoretical structural compositions was established to assist the mass spectra interpretation. The six digests derived by three enzymes from two types of LMWHs exhibited distinguishable fingerprinting patterns. And a total of 94 enoxaparin fragments and 109 nadroparin fragments were detected and identified. Besides the common LMWH oligosaccharides, many components containing characteristic LMWH structures such as saturated L-idopyranosuronic acid, 2,5-anhydro-D-mannitol, 1,6-anhydro-D-aminopyranose, as well as odd number oligosaccharides were also revealed. Quantitative comparison of major components derived from innovator and generic nadroparin products was presented. This approach to profile LMWHs' fragments offers a highly reproducible, high resolution and information-rich tool for evaluating the quality of this category of anticoagulant drugs or comparing structural similarities among samples from various sources.
低分子量肝素（LMWHs）是通过肝素的化学或酶解聚制备的线性且带高电荷的碳水化合物聚合物。与普通肝素（UFH）相比，LMWHs因其副作用较低和生物利用度较好而被广泛用作临床抗凝药物。本文介绍的工作为LMWHs的结构表征提供了一种快速且强大的片段图谱绘制方法。通过用肝素酶I（Hep I，酶委员会（EC）编号# 4.2.2.7）、肝素酶II（Hep II，未指定EC编号）和肝素酶III（Hep III，EC编号# 4.2.2.8）进行可控酶消化，生成了依诺肝素和那屈肝素两种LMWHs的链片段。采用反相离子对高效液相色谱（RPIP-HPLC）与电喷雾离子阱飞行时间质谱（ESI-IT-TOF-MS）联用，对从二糖到十糖的寡糖链进行分析。建立了一个包含所有理论结构组成的数据库，以辅助质谱解释。由三种酶从两种LMWHs衍生得到的六种消化产物呈现出可区分的指纹图谱模式。总共检测并鉴定出94个依诺肝素片段和109个那屈肝素片段。除了常见的LMWH寡糖外，还发现了许多含有特征性LMWH结构的成分，如饱和L-艾杜糖醛酸、2,5-脱水-D-甘露糖醇、1,6-脱水-D-氨基吡喃糖以及奇数寡糖。对创新型和仿制药那屈肝素产品的主要成分进行了定量比较。这种分析LMWHs片段的方法为评估这类抗凝药物的质量或比较不同来源样品的结构相似性提供了一种高度可重现、高分辨率且信息丰富的工具。
