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

Abstract

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.

Urinary Oxidative Stress Biomarkers in Workers of a Titanium Dioxide Based Pigment Production Plant

“…The methylation product of cytidine, 5-methylcytidine (5-MeCyt), has also been quantified as an epigenetic marker of RNA, and reduced levels of this modified nucleoside have been found to be associated with various tumors [11]. This oxidation process can also involve lipids and proteins: For proteins, it implies the introduction of functional groups that could alter their functionality and metabolism.…”

Quantification of DNA Lesions Induced by 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanol in Mammalian Cells

“…In this respect, methods including 32 P-postlabeling assay, excision assay, and immunoblot analysis have been used for measuring the levels and assessing the repair of targeted DNA lesions in vitro and in vivo. 31,32 Due to its high sensitivity and its capacity in providing structural information, 18,[33][34][35][36][37][38][39][40][41] mass spectrometry has become a tool of choice in DNA adduct analysis. In this vein, solid-phase extraction followed by LC-MS/MS analysis was previously used for quantifying several pyridyloxobutyl and pyridylhydroxybutyl DNA lesions.…”

Elevated Levels of Oxidative Nucleic Acid Modification Markers in Urine From Gastric Cancer Patients: Quantitative Analysis by Ultra Performance Liquid Chromatography-Tandem Mass Spectrometry

“…Although other analytical methods, e.g., enzyme-linked immunosorbent assay (ELISA), were utilized to analyze 8-OHdG and 8-OHG (Kobayashi et al, 2015 ; Sliwinska et al, 2016 ), the limited specificity which often leads to the overestimation of the concentrations is a major problem. As a powerful analytical tool, the stable isotope dilution LC-MS/MS technique can achieve high accuracy because the analyte loss during the pretreatment procedure and the ionization efficiency of analytes in mass spectrometer are compensated (Guo et al, 2018a ). In order to improve the separation efficiency and make the analysis time shorter, substitution of conventional high-performance liquid chromatography (HPLC) by ultra-performance liquid chromatography (UPLC) is a commonly used strategy.…”