一种改良的分枝菌酸提取及其非放射性TLC检测方法

An improved extraction and nonradioactive thin-layer chromatography detection method of mycolic acid

摘要:

分枝菌酸(mycolic acid, MA)是存在于分枝杆菌细胞壁中的独特长链脂肪酸，与分枝杆菌(mycobacterium)抵御不利环境、耐受抗生素和逃避宿主免疫密切相关，是较热门的抗结核药物筛选的靶点。MA的检测方法主要有放射性薄层层析(thin-layer chromatography, TLC)和液相色谱-质谱(liquid chromatograph mass spectrometer, LC-MS)，受放射性元素使用资质和标准品等的限制，MA分析是分枝杆菌相关研究的一个难点。本研究采用一种普通薄层层析技术，并对使用四丁基氢氧化铵溶液水解酯化的分枝菌酸，将其甲酯化后再用无水乙醚萃取分枝菌酸甲酯的操作步骤进行改良，使分枝杆菌的MA提取及分析可在常规生物实验室中开展。研究通过比较不同分枝杆菌及不同生长时期的MA成分与亚型特征、检测靶向分枝菌酸合成通路的抗结核药物对细菌MA合成影响以及分枝杆菌突变株对结核分枝杆菌(Mycobacterium tuberculosis, Mtb) H37Ra分枝菌酸合成影响等基础和应用研究的3个方面，进一步验证该方法在分枝杆菌MA分析中的实用性。结果表明该方法在不使用放射性元素和缺少标准品情况下可简便快速地分析MA及亚型特征，可广泛用于新型抗分枝杆菌药物筛选靶向分枝菌酸合成通路机制及基础研究中MA相关的分析和应用。

Abstract:

Mycolic acids (MAs), i.e. 2-alkyl, 3-hydroxy long-chain fatty acids, are the hallmark of the cell envelope of Mycobacterium tuberculosis and are related with antibiotic resistance and host immune escape. Nowadays, they’ve become hot target of new anti-tuberculosis drugs. There are two main methods to detect MAs, ¹⁴C metabolic labeling thin-layer chromatography (TLC) and liquid chromatograph mass spectrometer (LC-MS). However, the user qualification of ¹⁴C or the lack of standards for LC-MS hampered the easy use of this method. TLC is a common way to analyze chemical substance and can be used to analyze MAs. In this study, we used tetrabutylammonium hydroxide and methyl iodide to hydrolyze and formylate MAs from mycobacterium cell wall. Subsequently, we used diethyl ether to extract methyl mycolate. By this method, we can easily extract and analyze MA in regular biological labs. The results demonstrated that this method could be used to compare MAs of different mycobacterium in different growth phases, MAs of mycobacteria treated by anti-tuberculosis drugs or MAs of mycobacterium mutants. Therefore, we can use this method as an initial validation for the changes of MAs in researches such as new drug screening without using radioisotope or when the standards are not available.