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. 2013 May;59(5):824-32.
doi: 10.1373/clinchem.2012.193938. Epub 2013 Jan 23.

Quantification of 5-methylcytosine and 5-hydroxymethylcytosine in genomic DNA from hepatocellular carcinoma tissues by capillary hydrophilic-interaction liquid chromatography/quadrupole TOF mass spectrometry

Ming-Luan Chen  1 , Fan Shen, Wei Huang, Jia-Hui Qi, Yinsheng Wang, Yu-Qi Feng, Song-Mei Liu, Bi-Feng Yuan
Affiliations

Quantification of 5-methylcytosine and 5-hydroxymethylcytosine in genomic DNA from hepatocellular carcinoma tissues by capillary hydrophilic-interaction liquid chromatography/quadrupole TOF mass spectrometry

Ming-Luan Chen et al. Clin Chem. 2013 May.

Abstract

Background: 5-Methylcytosine (5-mC) is an important epigenetic modification involved in development and is frequently altered in cancer. 5-mC can be enzymatically converted to 5-hydroxymethylcytosine (5-hmC). 5-hmC modifications are known to be prevalent in DNA of embryonic stem cells and neurons, but the distribution of 5-hmC in human liver tumor and matched control tissues has not been rigorously explored.

Methods: We developed an online trapping/capillary hydrophilic-interaction liquid chromatography (cHILIC)/in-source fragmentation/tandem mass spectrometry system for quantifying 5-mC and 5-hmC in genomic DNA from hepatocellular carcinoma (HCC) tumor tissues and relevant tumor adjacent tissues. A polymer-based hydrophilic monolithic column was prepared and used for the separation of 12 nucleosides by cHILIC coupled with an online trapping system. Limits of detection and quantification, recovery, and imprecision of the method were determined.

Results: Limits of detection for 5-mC and 5-hmC were 0.06 and 0.19 fmol, respectively. The imprecision and recovery of the method were determined, with the relative SDs and relative errors being <14.9% and 15.8%, respectively. HCC tumor tissues had a 4- to 5-fold lower 5-hmC content compared to tumor-adjacent tissues. In addition, 5-hmC content highly correlated with tumor stage (tumor-nodes-metastasis, P = 0.0002; Barcelona Clinic liver cancer, P = 0.0003).

Conclusions: The marked depletion of 5-hmC may have profound effects on epigenetic regulation in HCC and could be a potential biomarker for the early detection and prognosis of HCC.

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Conflict of interest statement

Authors’ Disclosuresor Potential Conflicts of Interest: Upon manuscript submission, all authors completed the author disclosure form. Disclosures and/or potential conflicts of interest:

Figures

Fig. 1
Fig. 1. Experimental setup for the analysis of nucleosides (5-hmdC, 5-mdC, dC, dG, dA, dI, T, C, G, A, U, and I) by cHILIC-ESI-Q-TOF-MS
Fig. 2
Fig. 2. Extracted-ion chromatograms of nucleosides
(A), Nucleoside standards obtained under the optimized conditions. (B), Nucleosides from 2 ng genomic DNA of HCC tissues. Shown in the inset is the expanded chromatogram to reveal better the separation of 5-mdC, dC, C, dG, and 5-hmdC.
Fig. 3
Fig. 3. Quantification and statistical analysis of 5-mC and 5-hmC in human HCC tumor tissues and tumor-adjacent tissues
(A), 5-mC content in HCC tumor tissues and tumor-adjacent tissues. (B), 5-hmC content in HCC tumor tissues and tumor-adjacent tissues. (C), 5-mC content in matched-pair HCC tumor tissues and tumor-adjacent tissues. (D), 5-hmC content in matched-pair HCC tumor tissues and tumor-adjacent tissues. (E), Correlation of 5-hmC content with human HCC tumor TNM stages. (F), Correlation of 5-hmC content with human HCC tumor BCLC stages. (G), ROC curve for 5-hmC score for human HCC tumor tissues. (H), ROC curve for 5-mC score for human HCC tumor tissues. AUC [mean (SD)] are shown.
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