| PurposeThe objective of this study was to find non-invasive biomarkers for early diagnosis of colorectal cancer(CRC)by quantifying the modified nucleosides including 5-methyl-2'-deoxycytidine(5-mdC),5-hydroxymethyl-2'-deoxycytidine(5-hmdC),5-methylcytidine(5-mrC),5-hydroxymethylcytidine(5-hmrC)and 8-hydroxyguanosine(8-OHG)in human urine by ultra performance liquid chromatography tandem mass spectrometry(UPLC-MS/MS)and analyzing the correlation with colorectal cancer.MethodsThe study collected healthy controls' urine samples andCRC patients' urine samples from The Second Affiliated Hospital,Zhejiang University School of Medicine.After solid phase extraction(SPE)of the collected samples were performed,and the contents of 5-mdC,5-hmdC,5-mrC,5-hmrC,and 8-OHG in the urine were determined by the established UPLC-MS/MS methods,and the creatinine(Cr)levels acted as the correction factor for the urine concentrations.The corrected results were analyzed to find the correlation between the levels of modified nucleosides and colorectal cancer.ResultsFirstly,the levels of 8-OHG in the urine of CRC patients were significantly higher than those in the urine of healthy volunteers(P < 0.0001);the levels of 8-OHG in the urine of CRC patients aged older than 65 years were significantly higher than those in the urine of younger patients(P < 0.01).Gender and smoking history made no effect on the contents of 8-OHG in human urine(P > 0.05).Secondly,the levels of 5-mdC,5-hmdC,5-mrC,and 5-hmrC in the urine of CRC patients were significantly lower than those in the urine of healthy volunteers(P < 0.05),and the levels of 5-hmrC in the urine of men were significantly lower than those in the urine of women(P < 0.05).The levels of 5-hmrC in the urine of CRC patients with smoking history were significantly lower than those in the urine of non-smokers(P < 0.05).ConclusionsIn this study,the accurate,sensitive,stable and rapid UPLC-MS/MS methods were used to quantitatively analyze the contents of 5-mdC,5-hmdC,5-mrC,5-hmrC,and 8-OHG in human urine,and those correlations with colorectal cancer were discussed.The urinary 5-mdC,5-hmdC,5-mrC,and 5-hmrC contents in CRC patients were significant lower than those of the healthy controls,while the urinary 8-OHG contents in CRC patients were significant higher than those of the healthy controls,suggesting that those modifications could act as potential noninvasive biomarkers for the early discernment of CRC.In addition,the change of these oxidized,methylated and hydromethylated nucleosides in urine may be a common feature of CRC,which will help to study the effects of oxidative stress,methylation and demethylation on the formation and progression of colorectal cancer. |
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