LC-MS/MS测定血液全基因组DNA甲基化及其在糖尿病神经病变患者体内的应用

Determination of global DNA methylation in diabetic neuropathy blood by LC-MS/MS and its application

目的建立液相色谱-串联色谱法同时测定5-甲基胞嘧啶(5-mCyt)和胞嘧啶(Cyt)的浓度,初步探索糖尿病神经病变(DPN)患者全基因组DNA甲基化水平。方法糖尿病神经病变患者血液样品经HiPure Blood DNA Kits试剂盒提取DNA,酸水解DNA形成单个核酸,采用LC-MS/MS法定量测定核酸含量。测定条件色谱柱为WATERS Xterra■ RP18(4.6 mm×100 mm,3.5μm),流动相为甲醇-水梯度洗脱,电喷雾离子源正离子检测,三重四极杆质谱仪进行多反应监测模式。考察该方法的标准曲线、精密度、基质效应、稳定性。结果5-mCyt和Cyt水溶液在1~500 ng·mL^-1浓度范围内线性关系良好,批内、批间精密度均在15%以内,相对偏差在±15%,样本于-80℃放置2个月稳定性良好。临床预试验中,中度和重度DPN患者的DNA甲基化率为(4.0±1.2)%,轻度DPN患者DNA甲基化率为(5.0±1.2)%。结论本方法适用于血液样本中全基因组DNA甲基化的检测,DPN严重程度较重患者的DNA甲基化率较轻度DPN患者的甲基化率低。

Objective The aim of the study was to establish a liquid chromatography-tandem mass spectrometric method for the determination of the global DNA methylation level in diabetic neuropathy(DPN) blood. Methods DNA was extracted from plasma samples with HiPure Blood DNA Kits and then hydrolyzed by acid into single nucleic acid. The separation of nucleic acids was achieved on a WATERS Xterra■ RP18 column(4.6 mm×100 mm, 3.5 μm) with a mobile phase consisting of methanol and water under gradient elution. Electrospray ionization source was applied and operated in the positive multiple reaction monitoring mode for the quantification of 5-methylcytosine(5-mCyt) and cytosine(Cyt). The specificity, calibration, lower limit of quantification, precision, recovery and stability was evaluated. Results The linear relationship was good within 1~500 ng·mL-1 of both nucleic acids in water solution. The inter-and intra-batch precision were less than 15 % with relative error within ±15%. Both nucleic acids were stable enough under-80 ℃ for two months. The global DNA methylation ratio of severe DPN was less than mild DPN [(4.0±1.2)% vs(5.0±1.2)%]. Conclusion The method is accurate,sensitive,simple and suitable for the determination of global DNA methylation.