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Analytical Cellular Pathology
Volume 36 (2013), Issue 3-4, Pages 71-83

Absolute Quantification of Choline-Related Biomarkers in Breast Cancer Biopsies by Liquid Chromatography Electrospray Ionization Mass Spectrometry

Maria Chiara Mimmi,1 Nicoletta Finato,2 Gloria Pizzolato,1 Carlo Alberto Beltrami,2 Federico Fogolari,1 Alessandra Corazza,1 and Gennaro Esposito1

1Department of Medical and Biological Sciences, University of Udine, Piazzale M. Kolbe, Udine, Italy
2Department of Pathology, University of Udine, Piazzale Santa Maria della Misericordia, Udine, Italy

Copyright © 2013 Hindawi Publishing Corporation and the authors. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.


It has been repeatedly demonstrated that choline metabolism is altered in a wide variety of cancers. In breast tumours, the choline metabolite profile is characterized by an elevation of phosphocholine and total choline-compounds. This pattern is increasingly being exploited as biomarker in cancer diagnosis.

The majority of in vitro metabolomics studies, for biomarkers quantification in cell cultures or tissues, entail proton NMR spectroscopy. Although many “targeted” approaches have been proposed to quantify metabolites from standard one-dimensional (1D) NMR experiments, the task is often made difficult by the high degree of overlap characterizing 1H NMR spectra of biological samples.

Here we present an optimized protocol for tissue extraction and absolute quantification of choline, phosphocholine and glycerophosphocholine by means of liquid chromatography electrospray ionization mass spectrometry (LC-ESI-MS). The selected chromatographic separation system with a HILIC (hydrophilic interaction chromatography) amide column effectively separates free choline and its phopshorylated derivatives, contrary to failure observed using standard reversed-phase chromatography. The metabolite absolute quantification is based on external calibration with commercial standards, and is validated by a parallel 1D proton NMR analysis.

The LC-MS/NMR analysis is applied to three breast carcinoma specimens obtained by surgical excision, each one accompanied by a control tissue sample taken outside the tumor margin. The metabolite concentrations measured are in good agreement with previous results on metabolic profile changes of breast cancer. Each of the three cancerous biopsies, when compared with the control tissue, exhibit a highly increased levels phosphocholine, total choline and phosphocholine/glycerophosphocholine ratio.