测定野生型肝脏和牛奶样品中的唾液酸<em> CMAH</em>敲出小鼠
Summary
我们描述了一种基于HPLC的方法，用于测定小鼠肝脏和牛奶中的N-乙酰神经氨酸和N-羟乙酰神经氨酸。
Abstract
CMAH（胞苷一磷酸-N-乙酰神经氨酸羟化酶）负责哺乳动物中胞苷一磷酸-N-乙酰神经氨酸的氧化。然而，由于CMAH基因的主要外显子缺失，人类不能将胞苷一磷酸-N-乙酰神经氨酸氧化为胞苷一磷酸-N-羟乙酰神经氨酸。为了更清楚地了解CMAH活动缺乏的影响和影响，小鼠中的Cmah敲除模型对基础和应用研究非常感兴趣。本文详细描述了确定该小鼠模型的表型的分析方法，并且基于在野生型和Cmah敲除小鼠的肝脏和牛奶中检测到N-乙酰神经氨酸和N-羟乙酰神经氨酸。内源性唾液酸被邻苯二胺释放并衍生，产生荧光衍生物，随后可用HPLC分析。所呈现的协议可以是也适用于各种其他来源的牛奶和组织样品的分析，可用于研究N-羟乙酰神经氨酸的营养和健康影响。
Introduction
N-乙酰神经氨酸（Neu5Ac）和N-羟乙酰神经氨酸（Neu5Gc）是大多数哺乳动物中最常见的唾液酸1 。虽然能够内源性合成的Neu5Ac，人类不能产生Neu5Gc的由于对CMAH编码用于CMP-Neu5Ac的羟化酶2,3该基因的初级外显子缺失。然而，动物类食品产品可以Neu5Gc的4，5，6的饮食来源，导致产生抗Neu5Gc抗体，并因此触发朝向的Neu5Gc 7的免疫应答。 Neu5Gc的这一膳食效果被怀疑向慢性炎症和各种其它疾病8，9，10。为了全面了解Neu5Gc在h中的作用umans，一种用于系统研究食源性唾液酸影响的动物模型是非常需要的。虽然基于聚合酶链反应（PCR）分析敲除小鼠的方案已经很好地建立，并且是基因型评估的便捷方式，但代谢水平上表型的功能分析需要更具体的分析方法。可以通过分离和分析肝脏或牛奶样品中唾液酸的组成来评估Cmah敲除小鼠模型的表型。以前已经报道了几种用于检测动物组织中唾液酸的方法：唾液酸与间苯二酚11或硫代巴比妥酸12反应导致形成发色产物，并且可以使用基于平板印刷机的设置进行简单的分析，但只有总唾液酸可以测定酸含量。或者，也使用气相色谱法描述唾液酸的分析13 ，MALDI-ToF质谱法14或电流法15 。然而，最常用的唾液酸分析方法是基于水解释放，随后荧光衍生化和随后的高效液相色谱16，17，18。
Protocol
涉及动物受试者的程序已经根据“国家实验动物福利准则”（中国科学技术部，2006年），南京农业大学实验动物中心伦理委员会批准，动物饲养在SPF设施（Permission ID：SYXK-J-2011-0037）。 Cmah敲出鼠标模型使用扬州大学比较医学中心野生型C57Bl / 6小鼠（中国）。 注意：基于商业上使用CRISPR / Cas9 20战略来自先前CMAH敲除研究<sup class="…
Representative Results
所描述的分析方法的示意图如图1所示，包括从野生型和Cmah敲除突变小鼠的牛奶和肝脏样品中分离唾液酸，以及这些组分的荧光衍生化和HPLC分析。 图2和图3显示了来自同源和杂合敲除Cmah小鼠（ – / – 和+/-）和野生型小鼠的牛奶和肝脏样品的衍生唾液酸的代表性HPLC色谱图</strong…
Discussion
本文提出的方案允许通过分析和定量牛奶和肝脏样品的Neu5Gc的相对量来对纯合Cmah敲除小鼠进行表型评估。使用具有荧光检测的标准HPLC装置进行分析。该方法最关键的步骤是制备阴离子交换柱并进行阴离子交换层析;妥善安置树脂并收集正确的洗涤和洗脱部分需要一些练习。
或者，本文使用的衍生化试剂OPD可以容易地用更昂贵的衍生剂DMB（1,2-二氨基-4,5-亚甲基二氧基苯?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项工作部分得到了中国自然科学基金资助（授权号31471703，合资和律师事务所A0201300537和31671854）以及100名外籍人才计划（授予合资公司JSB2014012）。
Materials
| Chemicals: | |||
| N-acetylneuraminic acid | Sigma | A0812 | |
| N-glycolylneuraminic acid | Sigma | 50644 | 1 mg aliquot should be sufficient |
| o-Phenylenediamine | Sigma | 694975 | |
| Sodium hydrogen sulfite | J&K Scientific Ltd | 75234 | |
| Tools/Materials: | |||
| 3 mL SPE tubes | Supelco | Sigma 57024 | empty solid phase extraction columns |
| Luer stopcock | Sigma | S7396 | to stop the flow of the SPE tube |
| Dowex 1X8 | Dow Chemicals | Sigma 44340 | 200-400 mesh |
| Dounce tissue grinder | Sigma | D8938 | tight fit |
| HPLC Analysis: | |||
| High-recovery HPLC vial | Agilent Technologies | #5188-2788 | |
| HPLC System | Shimadzu | Nexera | |
| Fluorescence Detector for HPLC | Shimadzu | RF-20Axs | |
| HPLC Column | Phenomenex | Hyperclone ODS | 250×4.6 mm |
| LCMS-grade H2O | Merck Millipore | #WX00011 | |
| LCMS-grade Acetonitrile | Merck Millipore | #100029 | Hypergrade |
| Ammonium hydroxide solution | Fluka | #44273 | puriss. P.a. |
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Cite This Article
Cao, C., Wang, W. J., Huang, Y. Y., Yao, H. L., Conway, L. P., Liu, L., Voglmeir, J. Determination of Sialic Acids in Liver and Milk Samples of Wild-type and CMAH Knock-out Mice.. J. Vis. Exp. (125), e56030, doi:10.3791/56030 (2017).