A novel malic acid-enhanced method for the analysis of 5-methyl-2'-deoxycytidine, 5-hydroxymethyl-2'-deoxycytidine, 5-methylcytidine and 5-hydroxymethylcytidine in human urine using hydrophilic interaction liquid chromatography-tandem mass spectrometry.
A novel malic acid-enhanced method for the analysis of 5-methyl-2'-deoxycytidine, 5-hydroxymethyl-2'-deoxycytidine, 5-methylcytidine and 5-hydroxymethylcytidine in human urine using hydrophilic interaction liquid chromatography-tandem mass spectrometry.
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一种新的苹果酸增强方法，采用亲水相互作用液相色谱-串联质谱法分析人尿液中的 5-甲基-2'-脱氧胞苷、5-羟甲基-2'-脱氧胞苷、5-甲基胞苷和 5-羟甲基胞苷。
DOI：
10.1016/j.aca.2018.06.081
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发表时间：
2018-11-30
影响因子：
6.2
通讯作者：
Wang Y
中科院分区：
文献类型：
--
作者：
Guo C;Xie C;Chen Q;Cao X;Guo M;Zheng S;Wang Y
5-甲基-2 ′-脱氧胞苷（5-mdC）、5-羟甲基-2 ′-脱氧胞苷（5-hmdC）、5-甲基胞苷（5-mrC）和5-羟甲基胞苷（5-hmrC）是DNA和RNA的表观遗传标记，这些修饰的核苷的异常水平被发现与多种癌症有关。尿液是用于生物标志物发现的生物流体的优选来源，因为样品收集过程对患者没有侵入性。在此，我们开发了一种新的苹果酸增强的亲水相互作用液相色谱-串联质谱（HILIC-MS/MS）方法，用于灵敏和同时定量的人尿液中的修饰胞嘧啶核苷。苹果酸显著提高了所有四种胞嘧啶核苷的检测灵敏度，5-mdC，5-hmdC，5-mrC和5-hmrC的检测限（LOD）分别为0.025，0.025，0.025和0.050 fmol。通过使用这种方法，我们首次证明了5-hmrC在人尿液中的存在，并且我们成功地定量了90例结直肠癌（CRC）患者和90例健康对照者的尿液样品中的5-mdC、5-hmdC、5-mrC和5-hmrC。我们发现，大肠癌患者尿液中5-mdC、5-hmdC、5-mrC和5-苹果酸大肠癌hmrC的水平均显著降低，这表明这些修饰的核苷可能具有很大的潜力，可作为大肠癌早期检测和预后的非侵入性生物标志物。我们建立了一种新的检测5-甲基化和5-甲基化的灵敏方法，本研究的结果可能会刺激未来的研究，这些胞嘧啶衍生物在CRC的发生和发展中的调节作用。
5-Methyl-2′-deoxycytidine (5-mdC), 5-hydroxymethyl-2′-deoxycytidine (5-hmdC), 5-methylcytidine (5-mrC) and 5-hydroxymethylcytidine (5-hmrC) are epigenetic marks of DNA and RNA, and aberrant levels of these modified nucleosides were found to be associated with various cancers. Urine is a preferred source of biological fluid for biomarker discovery because the sample collection process is not invasive to patients. Herein, we developed a novel malic acid-enhanced hydrophilic interaction liquid chromatography-tandem mass spectrometry (HILIC-MS/MS) method for sensitive and simultaneous quantification of the modified cytosine nucleosides in human urine. Malic acid markedly increased the detection sensitivities of all four cytosine nucleosides, with the limits of detection (LODs) for 5-mdC, 5-hmdC, 5-mrC and 5-hmrC being 0.025, 0.025, 0.025 and 0.050 fmol, respectively. By using this method, we demonstrated, for the first time, the presence of 5-hmrC in human urine, and we successfully quantified 5-mdC, 5-hmdC, 5-mrC and 5-hmrC in urine samples collected from 90 patients with colorectal cancer (CRC) and 90 healthy controls. We found that the levels of 5-mdC, 5-hmdC, 5-mrC and 5-Malic acid Colorectal cancer hmrC in urine were all substantially decreased in CRC patients, suggesting that these modified nucleo-sides might have great potential to be noninvasive biomarkers for early detection and prognosis of CRC.Together, we established a novel and sensitive method for detecting 5-methylated and 5-hydroxymethylated cytosine nucleosides in human urine and the results from this study may stimulate future investigations about the regulatory roles of these cytosine derivatives in the initiation and development of CRC.
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