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Clinical Chemistry and Laboratory Medicine (CCLM)

Published in Association with the European Federation of Clinical Chemistry and Laboratory Medicine (EFLM)

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Ed. by Gillery, Philippe / Lackner, Karl J. / Lippi, Giuseppe / Melichar, Bohuslav / Payne, Deborah A. / Schlattmann, Peter / Tate, Jillian R.

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Global DNA methylation measured by liquid chromatography-tandem mass spectrometry: analytical technique, reference values and determinants in healthy subjects

Robert M. Kok1 / Desirée E.C. Smith2 / Rob Barto3 / Annemieke M.W. Spijkerman4 / Tom Teerlink5 / Henk J. Gellekink6 / Cornelis Jakobs7 / Yvo M. Smulders8

1Department of Clinical Chemistry, VU University Medical Center, Amsterdam, The Netherlands

2Department of Clinical Chemistry, VU University Medical Center, Amsterdam, The Netherlands

3Department of Clinical Chemistry, VU University Medical Center, Amsterdam, The Netherlands

4Institute for Research in Extramural Medicine, VU University Medical Center, Amsterdam, The Netherlands

5Department of Clinical Chemistry, VU University Medical Center, Amsterdam, The Netherlands

6Department of Clinical Chemistry, Radboud University Nijmegen Medical Center, Nijmegen, The Netherlands

7Department of Clinical Chemistry, VU University Medical Center, Amsterdam, The Netherlands

8Department of Internal Medicine and Institute for Cardiovascular Research ICaR-VU, VU University Medical Center, The Netherlands

Corresponding author: Yvo M. Smulders, MD, VU University Medical Center, Department of Internal Medicine, P.O. Box 7057, Amsterdam 1007 MB, The Netherlands Phone: +31-20-4444307, Fax: +31-20-4444313

Citation Information: Clinical Chemical Laboratory Medicine. Volume 45, Issue 7, Pages 903–911, ISSN (Online) 14374331, ISSN (Print) 14346621, DOI: 10.1515/CCLM.2007.137, July 2007

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Published Online:


Background: Alterations in global DNA methylation are implicated in various pathobiological processes. We describe a liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) method for determination of cytosine and 5-methylcytosine in DNA.

Methods: DNA was hydrolyzed using formic acid. Cytosine and 5-methylcytosine were separated by gradient-elution reversed-phase chromatography with a mobile phase containing nonafluoropentanoic acid (NFPA) as ion-pairing reagent and quantified using stable isotope dilution LC-ESI-MS/MS. The method was applied to DNA isolated from leukocytes of healthy volunteers.

Results: Linear calibration curves were obtained in the range 0.111–4.422 ng/μL [mean correlation co-efficient 0.9983 (SD=0.0011), n=9] for cytosine and 0.0048–0.1936 ng/μL [mean correlation coefficient 0.9991 (SD=0.0010), n=9] for 5-methylcytosine. The intra- and inter-assay CVs for the 5-methylcytosine/total cytosine ratio (mCyt/tCyt) was 1.7% (n=9) and 3.5% (n=8) for calf thymus DNA (mean mCyt/tCyt ratio 6.5%), and 4.5% (n=6) and 6.5% (n=14), respectively for pBR322 DNA (mean mCyt/tCyt ratio 0.48%). The limit of detection (signal-to-noise ratio 3) was 2 pg on-column for cytosine and 5-methylcytosine. In healthy subjects (n=109), the mCyt/tCyt ratio varied from 2.6% to 4.8% (median 4.1%). DNA methylation was negatively correlated to age, but only in subjects with the methylenetetrahydrofolate reductase (MTHFR) 677 TT genotype (p=0.046). No association with B-vitamin status was observed.

Conclusions: This LC-ESI-MS/MS method is easy to perform and offers reproducibility, selectivity and sensitivity for studying DNA methylation. The method allows a sample throughput of approximately 200 samples/week. The MTHFR C677T genotype influences age-related changes in DNA methylation.

Clin Chem Lab Med 2007;45:903–11.

Keywords: cytosine; global DNA methylation; isotope dilution; 5-methylcytosine; methylenetetrahydrofolate reductase (MTHFR); tandem mass spectrometry

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