Abstract
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.
Highlights
Choline (Cho) is a dietary component essential for the normal function of all cells [11]
magnetic resonance spectroscopy (MRS) investigation on breast cancers recognised the occurrence of a switch from high glycerophosphocholine (GPCho) and low PCho to low GPCho and high PCho in presence of cancerogenesis
It should be considered that PCho alteration is not exclusive to malignancy, since it may occur in other cases of accelerated tissue proliferation like for instance benign tumours or developing organs
Summary
Choline (Cho) is a dietary component essential for the normal function of all cells [11]. The intracellular metabolism of choline in breast is partitioned among two major pathways: (a) synthesis of phosphatidylcholine (PtdCho) and (b) oxidation to produce the methyl donor betaine. The abnormal choline metabolite profile typical of cancers is characterized by an elevation of phosphocholine (PCho) and total choline (tCho) containing metabolites The major molecular causes of the increased PCho and tCho levels in cancer cells and tumors include an increased expression and activity of choline kinase alpha (CHKalpha), an increased activities of phosphatidylcholine-specific phospholipase C and D (PLC and PLD), and phospholipases A2 (PLA2) [5]. MRS investigation on breast cancers recognised the occurrence of a switch from high glycerophosphocholine (GPCho) and low PCho to low GPCho and high PCho in presence of cancerogenesis
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