Targeting amine- and phenol-containing metabolites in urine by dansylation isotope labeling and liquid chromatography mass spectrometry for evaluation of bladder cancer biomarkers

Yi-Ting Chen ^a,b,c,d,*, Hsin-Chien Huang ^b, Ya-Ju Hsieh ^b, Shu-Hsuan Fu ^b, Liang Li ^e, Chien-Lun Chen ^f,g, Lichieh Julie Chu ^b,h, Jau-Song Yu ^b,h,i,j

^a Graduate Institute of Biomedical Sciences, College of Medicine, Chang Gung University, Taoyuan, Taiwan

^b Molecular Medicine Research Center, College of Medicine, Chang Gung University, Taoyuan, Taiwan

^c Department of Biomedical Sciences, College of Medicine, Chang Gung University, Taoyuan, Taiwan

^d Department of Nephrology, Chang Gung Memorial Hospital, Linkou Medical Center, Taoyuan, Taiwan

^e Department of Chemistry, University of Alberta, Edmonton, AB, T6G2G2, Canada

^f Department of Urology, Chang Gung Memorial Hospital, Taoyuan, Taiwan

^g College of Medicine, Chang Gung University, Taoyuan, Taiwan

^h Liver Research Center, Chang Gung Memorial Hospital at Linkou, Gueishan, Taoyuan, 33305, Taiwan

ⁱ Department of Cell and Molecular Biology, Chang Gung University, Guishan, Taoyuan, 33302, Taiwan

^j Research Center for Food and Cosmetic Safety, Research Center for Chinese Herbal Medicine, College of Human

Ecology, Chang Gung University of Science and Technology, Taoyuan, Taiwan

Metabolomics is considered an effective approach for understanding metabolic responses in complex biological systems. Accordingly, it has attracted increasing attention for biomarker discovery, especially in cancer. In this study, we used a non-invasive method to evaluate four urine metabolite biomarker candidates-o-phosphoethanolamine, 3-amio-2-piperidone, uridine and 5-hydroxyindoleactic acid -for their potential as bladder cancer diagnostic biomarkers. To analyze these targeted amine- and phenol-containing metabolites, we used differential ¹²C₂-/¹³C₂-dansylation labeling coupled with liquid chromatography/ tandem mass spectrometry, which has previously been demonstrated to exhibit high sensitivity and reproducibility. Specifically, we used ultra-performance liquid chromatography (UPLC) coupled with high-resolution Fourier transform ion-cyclotron resonance MS system (LC-FT/MS) and an ion trap MS with MRM function (LC-HCT/MS) for targeted quantification. The urinary metabolites of interest were well separated and quantified using this approach. To apply this approach to clinical urine specimens, we spiked samples with ¹³C₂-dansylatedsynthetic compounds, which served as standards for targeted quantification of ¹²C₂-dansylated urinary endogenous metabolites using LC-FT/MS as well as LC-HCT/MS with MRM mode. These analyses revealed significant differences in two of the four metabolites of interest-o-phosphoethanolamine and uridine-between bladder cancer and non-cancer groups. O-phosphoethanolamine was the most promising single biomarker, with an area-under-the-curve (AUC) value of 0.709 for bladder cancer diagnosis. Diagnostic performance was improved by combining uridine and o-phosphoethanolamine in a marker panel, yielding an AUC value of 0.726. This study confirmed discovery-phase features of the urine metabolome of bladder cancer patients and verified their importance for further study.

Keywords: Bladder cancer, Biomarker, Phosphoethanolamine, Uridine, Metabolites