Clinical Chemistry and Laboratory Medicine (CCLM)
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Global DNA methylation measured by liquid chromatography-tandem mass spectrometry: analytical technique, reference values and determinants in healthy subjects
- 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
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.
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