Preliminary Investigation Of Urinary Biomarkers For Renal Cell Carcinoma Diagnosis Based On Metabonomics

Objective:Due to the lack of obvious clinical symptoms and the absence of typical humoral biomarkers for early diagnosis in patients with renal cell carcinoma（RCC）,about one third of patients with RCC were diagnosed as advanced renal cancer at the first visit,and the mortality rate was as high as 40%.Usually,some cancers are difficult to differentiate from clear cell carcinomas and easy to be misdiagnosed.Therefore,early diagnosis of renal cancer is extremely important.The urine samples were used from patients of RCC and healthy volunteers.Metabonomics based on nuclear magnetic resonance（¹H-NMR）and HPLC-HILIC-UV was employed to identify and validate the urinary metabolites that could distinguish between RCC and healthy people.Methods:1 Normal control group chose healthy people who volunteered to take part in physical examination（n=31）.In the experimental group,patients with RCC confirmed by histopathological diagnosis before operation（n=42）and patients after nephrectomy（n=26）were selected.Urine samples were pre-treated properly and used for ¹H-NMR analysis.SIMCA-P was used to analyze ¹H-NMR data.Using network databases,total correlation spectroscopy（TOCSY）,¹³C-heteronuclear single quantum correlation^{（13）C-HSQC},statistical total correlation spectroscopy（STOCSY）and SOM（SOM）E standard compounds are also used to identify metabolites.2 Quantitative determination of xanthine,hypoxanthine,uric acid and creatinine in urine before and after HS and RCC operation by HPLC-HILIC-UV.The required differential metabolites were selected as variables,and the corresponding ¹H-NMR spectra of all variables and the data collected by HPLC-HILIC-UV were used as binary stepwise logistic regression analysis to select the best biomarker combination for the diagnosis of renal cancer.The ROC curve was drawn to evaluate its diagnostic efficiency.Results:1 ¹H-NMR data analysis showed that there were significant differences among normal control group,pre-operative renal cell carcinoma group and post-operative renal cell carcinoma group,and the post-operative group tended to return to the normal group.Compared with the normal group,the levels of lysine,phenylalanine,fumaric acid,formic acid and levulinic acid in the renal cell carcinoma group increased before operation,while the levels of xanthine and valine decreased.Compared with the preoperative renal cell carcinoma group,the levels of xanthine and valine in the renal cell carcinoma group increased after operation,while the other markers decreased in the renal cell carcinoma group after operation,and basically returned to normal level.2 The quantitative results of HPLC-HILIC-UV showed that the concentration of uric acid and creatinine in urine of renal cell carcinoma before operation was significantly higher than that of normal control group.The concentrations of C_UA/C_HX,C_UA/C_X,C_X/C_HX,(C_UA+C_X)/C_HX also showed an upward trend,but C_X/C_Crr showed a downward trend.The same batch of samples after nephrectomy were analyzed by HPLC-HILIC-UV.The results showed that the concentration of uric acid and creatinine decreased significantly,and there was a tendency to return to normal level.There were also C_UA/C_HX,C_UA/C_X,C_X/C_HX,(C_UA+C_X)/C_HX tended to return to normal level,and C_X/Cr increased four times.3 Quantitative data analysis of nuclear magnetic resonance and chromatography showed that valine,C_UA/C_X and C_HX/C_Cr could differentiate between normal and pre-operative renal cancer patients;valine,choline and C_HX/C_Cr could be used as biomarkers to differentiate pre-and post-operative renal cancer patients;and valine,C_UA/C_X and C_HX/C_Cr could also differentiate Pre-RCC/Post-RCC+HS patients.After logistic regression analysis,the AUCs of the combined variables were 0.967,0.857and 0.913（P<0.01）.After logistic regression analysis,the sensitivity of the combined variables was 0.968,0.885 and 0.842,the specificity was 0.881,0.756 and0.833,and the critical values were 0.276,0.270 and 0.539,respectively.Conclusion:In this dissertation,the urine samples of healthy volunteers and patients with renal cancer before and after operation were systematically analyzed by the combination of ¹H-NMR-based metabolomics technology and quantitative analysis of HPLC-HILIC-UV.The results showed that some amino acids and purines metabolites changed significantly before and after operation.The combination of urinary valine and C_HX/C_Cr variables could be used as potential diagnostic indicators for renal cancer and post-operation kidney.The monitoring indicators of convalescence of cancer deserve further study.