4.7 Article

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

Journal

ANALYTICA CHIMICA ACTA
Volume 1034, Issue -, Pages 110-118

Publisher

ELSEVIER SCIENCE BV
DOI: 10.1016/j.aca.2018.06.081

Keywords

DNA and RNA modification; 5-Methylated and 5-hydroxymethylated cytosine nucleosides; Hydrophilic interaction liquid chromatography-tandem mass spectrometry; Malic acid; Colorectal cancer

Funding

  1. National Natural Science Foundation of China [21402172, 81472666]
  2. Analysis and Detection Foundation of Science and Technology Department of Zhejiang Province [2015C37030]
  3. National Institutes of Health [R01 CA210072]
  4. NATIONAL CANCER INSTITUTE [R01CA210072] Funding Source: NIH RePORTER

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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-hmrC in urine were all substantially decreased in CRC patients, suggesting that these modified nucleosides 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. (C) 2018 Elsevier B.V. All rights reserved.

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