Jump to ContentJump to Main Navigation
Show Summary Details
More options …

Clinical Chemistry and Laboratory Medicine (CCLM)

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

Editor-in-Chief: Plebani, Mario

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

12 Issues per year

IMPACT FACTOR 2016: 3.432

CiteScore 2016: 2.21

SCImago Journal Rank (SJR) 2016: 1.000
Source Normalized Impact per Paper (SNIP) 2016: 1.112

See all formats and pricing
More options …
Volume 45, Issue 7 (Jul 2007)


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

Robert M. Kok / Desirée E.C. Smith / Rob Barto / Annemieke M.W. Spijkerman
  • 4Institute for Research in Extramural Medicine, VU University Medical Center, Amsterdam, The Netherlands
  • Other articles by this author:
  • De Gruyter OnlineGoogle Scholar
/ Tom Teerlink / Henk J. Gellekink
  • 6Department of Clinical Chemistry, Radboud University Nijmegen Medical Center, Nijmegen, The Netherlands
  • Other articles by this author:
  • De Gruyter OnlineGoogle Scholar
/ Cornelis Jakobs / Yvo M. Smulders
  • 8Department of Internal Medicine and Institute for Cardiovascular Research ICaR-VU, VU University Medical Center, The Netherlands
  • Other articles by this author:
  • De Gruyter OnlineGoogle Scholar
Published Online: 2007-07-08 | DOI: https://doi.org/10.1515/CCLM.2007.137


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

About the article

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

Received: 2007-01-17

Accepted: 2007-02-07

Published Online: 2007-07-08

Published in Print: 2007-07-01

Citation Information: Clinical Chemical Laboratory Medicine, ISSN (Online) 14374331, ISSN (Print) 14346621, DOI: https://doi.org/10.1515/CCLM.2007.137.

Export Citation

Citing Articles

Here you can find all Crossref-listed publications in which this article is cited. If you would like to receive automatic email messages as soon as this article is cited in other publications, simply activate the “Citation Alert” on the top of this page.

Yunbao Pan, Guohong Liu, Fuling Zhou, Bojin Su, and Yirong Li
Clinical and Experimental Medicine, 2017
Jessica A. Kilgore, Xinlin Du, Lisa Melito, Shuguang Wei, Changguang Wang, Hang Gyeong Chin, Bruce Posner, Sriharsa Pradhan, Joseph M. Ready, and Noelle S. Williams
Journal of Biological Chemistry, 2013, Volume 288, Number 27, Page 19673
Olga A Efimova, Anna A Pendina, Andrei V Tikhonov, and Vladislav S Baranov
Ecological genetics, 2016, Volume 14, Number 4, Page 14
Alexander Semmler, Christian Frisch, Christiane Bleul, Desiree Smith, Laurent Bigler, Jean-Christophe Prost, Henk Blom, and Michael Linnebank
Seizure, 2017, Volume 46, Page 7
Richard J. Honeywell, Dzjemma Sarkisjan, Ietje Kathmann, Michael H. Kristensen, and Godefridus J. Peters
Nucleosides, Nucleotides and Nucleic Acids, 2016, Volume 35, Number 10-12, Page 652
Mónica Cappetta, María Berdasco, Jimena Hochmann, Carolina Bonilla, Mónica Sans, Pedro C Hidalgo, Nora Artagaveytia, Rick Kittles, Miguel Martínez, Manel Esteller, and Bernardo Bertoni
BMC Cancer, 2015, Volume 15, Number 1
Will D King, Janet E Ashbury, Sherryl A Taylor, M Yat Tse, Stephen C Pang, Jacob A Louw, and Stephen J Vanner
BMC Cancer, 2014, Volume 14, Number 1
Oliver D.K. Maddocks, Christiaan F. Labuschagne, Peter D. Adams, and Karen H. Vousden
Molecular Cell, 2016, Volume 61, Number 2, Page 210
Frances M. Nilsen, Benjamin B. Parrott, John A. Bowden, Brittany L. Kassim, Stephen E. Somerville, Teresa A. Bryan, Colleen E. Bryan, Ted R. Lange, J. Patrick Delaney, Arnold M. Brunell, Stephen E. Long, and Louis J. Guillette
Science of The Total Environment, 2016, Volume 545-546, Page 389
Jorke H. Kamstra, Marianne Løken, Peter Aleström, and Juliette Legler
Zebrafish, 2015, Volume 12, Number 3, Page 230
Alexander Semmler, Peter Heese, Birgit Stoffel-Wagner, Marc Muschler, Annemarie Heberlein, Laurent Bigler, Jean-Christophe Prost, Helge Frieling, Johannes Kornhuber, Markus Banger, Stefan Bleich, Thomas Hillemacher, and Michael Linnebank
Alcohol, 2015, Volume 49, Number 2, Page 97
G. S. Madugundu, J. Cadet, and J. R. Wagner
Nucleic Acids Research, 2014, Volume 42, Number 11, Page 7450
B. B. Parrott, J. A. Bowden, S. Kohno, J. A. Cloy-McCoy, M. D. Hale, J. T. Bangma, T. R. Rainwater, P. M. Wilkinson, J. R. Kucklick, and L. J. Guillette
Reproduction, 2014, Volume 147, Number 4, Page 503
Bi-Feng Yuan and Yu-Qi Feng
TrAC Trends in Analytical Chemistry, 2014, Volume 54, Page 24
Sajin Bae, Cornelia M Ulrich, Lynn B Bailey, Olga Malysheva, Elissa C Brown, David R Maneval, Marian L Neuhouser, Ting-Yuan David Cheng, Joshua W Miller, Yingye Zheng, Liren Xiao, Lifang Hou, Xiaoling Song, Katharina Buck, Shirley AA Beresford, and Marie A Caudill
Epigenetics, 2014, Volume 9, Number 3, Page 396
Ed Dudley and Liz Bond
Mass Spectrometry Reviews, 2014, Volume 33, Number 4, Page 302
Yoshiaki Yamagata, Pàl Szabó, David Szüts, Caroline Bacquet, Tamàs Arànyi, and Andras Paldi
Epigenetics, 2012, Volume 7, Number 2, Page 141
Audrey Y. Jung, Akke Botma, Carolien Lute, Henk J. Blom, Per M. Ueland, Gry Kvalheim, Øivind Midttun, Fokko Nagengast, Wilma Steegenga, and Ellen Kampman
Molecular Nutrition & Food Research, 2013, Volume 57, Number 4, Page 698
Roosmarijn Luttmer, Annemieke M. Spijkerman, Robert M. Kok, Carel Jakobs, Henk J. Blom, Erik H. Serne, Jacqueline M. Dekker, and Yvo M. Smulders
Obesity Research & Clinical Practice, 2013, Volume 7, Number 2, Page e106
Angelo Zinellu, Salvatore Sotgia, Valentina Murtas, Paolo Cossu-Rocca, Maria Rosaria Miglio, Maria Rosaria Muroni, Antonica Mura, Maria Gabriela Uras, Marcella Contini, Luca Deiana, and Ciriaco Carru
Analytical and Bioanalytical Chemistry, 2011, Volume 399, Number 3, Page 1181
Ill Yang, Marie C. Fortin, Jason R. Richardson, and Brian Buckley
Analytical Biochemistry, 2011, Volume 409, Number 1, Page 138
Anders M.B. Giessing and Finn Kirpekar
Journal of Proteomics, 2012, Volume 75, Number 12, Page 3434
Salvatore Sotgia, Ciriaco Carru, Flavia Franconi, Paolo Battista Fiori, Salvatorica Manca, Silvestro Pettinato, Stefania Magliona, Renzo Ginanneschi, Luca Deiana, and Angelo Zinellu
Journal of Chromatography A, 2008, Volume 1185, Number 1, Page 145
Olga J. G. Schiepers, Martin P. J. van Boxtel, Renate H. M. de Groot, Jelle Jolles, Frans J. Kok, Petra Verhoef, and Jane Durga
British Journal of Nutrition, 2012, Volume 107, Number 05, Page 744
Kazimierz Wrobel, Julio Alberto Landero Figueroa, Silvio Zaina, Gertrud Lund, and Katarzyna Wrobel
Journal of Chromatography B, 2010, Volume 878, Number 5-6, Page 609
Jatinderpal Sandhu, Balvinder Kaur, Christine Armstrong, Christopher J. Talbot, William P. Steward, Peter B. Farmer, and Rajinder Singh
Journal of Chromatography B, 2009, Volume 877, Number 20-21, Page 1957
Xiaoli Wang, Yongshan Suo, Ruichuan Yin, Heqing Shen, and Hailin Wang
Journal of Chromatography B, 2011, Volume 879, Number 19, Page 1647
Jun-jie Zhang, Lijian Zhang, Keyuan Zhou, Xiaoxia Ye, Chunan Liu, Liangtao Zhang, Jingxuan Kang, and Chun Cai
Analytical Biochemistry, 2011, Volume 413, Number 2, Page 164
Holger Cario, Desirée E.C. Smith, Henk Blom, Nenad Blau, Harald Bode, Karlheinz Holzmann, Ulrich Pannicke, Karl-Peter Hopfner, Eva-Maria Rump, Zuleya Ayric, Elisabeth Kohne, Klaus-Michael Debatin, Yvo Smulders, and Klaus Schwarz
The American Journal of Human Genetics, 2011, Volume 88, Number 2, Page 226
Thuc Le, Kee-Pyo Kim, Guoping Fan, and Kym F. Faull
Analytical Biochemistry, 2011, Volume 412, Number 2, Page 203
Monica S. Rocha, Rita Castro, Isabel Rivera, Robert M. Kok, Yvo M. Smulders, Cornelis Jakobs, Isabel Tavares de Almeida, and Henk J. Blom
Clinical Chemistry and Laboratory Medicine, 2010, Volume 48, Number 12
T. K. Walsh, J. A. Brisson, H. M. Robertson, K. Gordon, S. Jaubert-Possamai, D. Tagu, and O. R. Edwards
Insect Molecular Biology, 2010, Volume 19, Page 215
Salvatore Sotgia, Angelo Zinellu, Elisabetta Pisanu, Luciano Murgia, Gerard Aime Pinna, Leonardo Gaspa, Luca Deiana, and Ciriaco Carru
Analytical and Bioanalytical Chemistry, 2010, Volume 396, Number 8, Page 2937
Monika Hassel, Michael G. Cornelius, Jochen vom Brocke, and Heinz H. Schmeiser
ELECTROPHORESIS, 2010, Volume 31, Number 2, Page 299
Federica Rossella, Elisa Polledri, Valentina Bollati, Andrea Baccarelli, and Silvia Fustinoni
Rapid Communications in Mass Spectrometry, 2009, Volume 23, Number 17, Page 2637
Li Xiao, Juan-Sebastian Saldivar, Cuilan Zhou, Chengli Chen, Jia Zhang, Pierre Sirois, and Kai Li
Molecular Biotechnology, 2009, Volume 41, Number 2, Page 152
K. A. Burren, D. Savery, V. Massa, R. M. Kok, J. M. Scott, H. J. Blom, A. J. Copp, and N. D.E. Greene
Human Molecular Genetics, 2008, Volume 17, Number 23, Page 3675
P. W. B. Nanayakkara, J. C. Kiefte-de Jong, C. D. A. Stehouwer, F. J. van Ittersum, M. R. Olthof, R. M. Kok, H. J. Blom, C. van Guldener, P. M. ter Wee, and Y. M. Smulders
Nephrology Dialysis Transplantation, 2008, Volume 23, Number 8, Page 2586
Journal of Mass Spectrometry, 2008, Volume 43, Number 3, Page 409
Mirosława Z. Barciszewska, Anna Maria Barciszewska, and Suresh I. S. Rattan
Biogerontology, 2007, Volume 8, Number 6, Page 673

Comments (0)

Please log in or register to comment.
Log in