Analysis of urinary nucleosides as potential tumor markers in human breast cancer by high performance liquid chromatography/electrospray ionization tandem mass spectrometry

Abstract

Background

Breast cancer is the most common female cancer and the fourth leading cause of cancer death among women in Taiwan. We measured urinary nucleoside levels in female breast cancer patients (n = 36) to evaluate the diagnostic value of nucleosides as potential tumor markers.

Methods

Purification of urinary nucleosides was performed using a 96-well solid phase extraction (SPE, cation-exchange column) procedure to decrease the variation between the single column preparations and to shorten the pretreatment time. Cation-exchange allows for the comprehensive purification of modified nucleosides, such as 2-deoxynucleosides, that are not purifiable by phenylboronic acid-based SPE. High-performance liquid chromatography (HPLC) coupled with mass spectrometry (MS) in selected reaction monitoring (SRM) mode was used to quantify multiple nucleosides. Tubercidin was used as an internal standard. The qualitative parameters, retention time, and the parent and daughter ions used revealed that the method was more specific and sensitive than traditional UV detection.

Results

Urinary levels of 3 nucleosides, cytidine, 3-methylcytidine, and inosine were significantly higher in breast cancer patients than in normal controls (p < 0.01). The discriminative powers of cytidine, 3-methylcytidine, and inosine were 58%, 58%, and 62%, respectively.

Conclusions

LC/MS/MS is a highly specific and sensitive method for rapidly screening a large number of urinary nucleosides that may be potential cancer markers. The 3-methylcytidine may be a candidate marker for breast cancer.

Introduction

The emerging field of metabolomics is characterized by the study of metabolic intermediates, hormones, signaling molecules, and secondary metabolites. In any biological system, metabolites of high chemical diversity are present in a dynamic range of concentrations that can exceed 10 orders of magnitude, thereby making quantitative analysis extremely challenging (Human Metabolome Database).

Mass spectrometry (MS) is a powerful tool in the field of metabolomics. There are two approaches to using MS when searching for metabolic biomarkers. In the untargeted approach, high-resolution mass spectrometry is usually used. By combining accurate mass analysis and fragmentation patterns, the structures of the metabolites can be assigned with relative certainty [1], [2], [3]. Statistical analyses can then be applied to the results of MS data to find significant differences in candidate biomarkers between disease and control groups. In the targeted approach, however, the candidate biomarkers are selected in advance to evaluate the discriminative power relative to the specific disease.

Nucleosides in urine are an important class of metabolites and have the potential of serving as tumor markers [4]. Modified nucleosides, regarded as indicators for the whole-body turnover of RNAs, are formed at the posttranscriptional stage by chemical modification of normal nucleosides. Modified nucleosides are excreted in abnormal amounts in the urine of patients with malignancies and several studies have shown a positive relation between nucleoside levels and cancer status [4], [5], [6]. Traditionally, analysis of urinary nucleosides has involved the use of liquid chromatography with ultraviolet detection (LC–UV) [7]. By comparing the chromatographic retention and UV spectra with known standard references, the nucleosides can be identified and quantified. When performing multiple analyses of nucleosides in urine, however, a time-consuming separation step is needed to avoid coelution and erroneous identification of large similar compounds. In contrast, MS is a more efficient technique for analyzing urinary nucleosides. Many novel nucleosides have been identified using MS and tandem MS coupled with liquid chromatography [1], [2], [3] or capillary electrophoresis [8] in untargeted analysis. Furthermore, both MS and tandem MS allow for the specific detection of coeluted nucleosides as long as they are different in parent or daughter mass.

Preliminary purification of nucleosides is a very important step prior to instrumental analysis. In our experience, the matrix suppression effect of urine causes poor ionization, especially of less hydrophobic nucleosides. Solid phase extraction (SPE) is a common method in sample preparation and phenylboronic acid (PBA) is widely used as the stationary phase because of its affinity for the vicinal hydroxyl group contained in the structure of nucleosides. PBA, however, is not effective at purifying deoxynucleoside structures such as 8-hydroxy-2-deoxyguanosine and 5-hydroxymethyl-deoxyuridine, two nucleosides that have been shown to be potential markers of breast cancer [9], [10], [11].