Study On Biomarkers Of Adverse Reactions Of Capecitabine Based On Targeted Bile Acid Metabolomics

Objective:To explore biomarkers of capecitabine adverse reactions using targeted bile acid omics.Methods:The Agilent 6460A triple quadrupole mass spectrometer equipped with an electrospray ionization source was used to simultaneously monitor 41 bile acids in negative ion mode using DMRM scanning method;and 41 bile acids were separated by gradient elution on Xbridge BEH C₁₈（2.1×75 mm,2.5μm,Waters,USA）column and methanol and water（containing 0.01%formic acid）as mobile phases.Sample pretreatment:First,the protein was precipitated with methanol,and the supernatant was evaporated under nitrogen.The residue was redissolved with methanol;Then,after vortex centrifugation,the supernatant was taken for analysis.The established method was verified according to the relevant requirements of Chinese pharmacopoeia（2015 edition）,and clinical samples were collected and clinical information（including demographic characteristics,medication cycle,reactions,etc）was recorded.The mouse models of hand-foot syndrome and diarrhea,was induced by intragastric administration of capecitabine（275 mg/kg,twice a day）for 14consecutive days.The successful establishment of the animal model was verified by HE staining,characteristic changes of foot plantar,and changes in fecal color/morphology.Mouse samples were collected.The data were processed by metaboanalyst 5.0 database,SIMCA 14.1 software and SPSS 19.0 software,and the correlation between bile acid concentration and adverse reactions of capecitabine was analyzed.Results:The established method can achieved within 10 min for 41 bile acids chromatographic separation and the peak shape is good.Methodological verification results meet the requirements of biological sample analysis,which can meet the rapid and high-throughput analysis of 41 kinds of bile acids in biological plasma samples.Results of animal model establishment:The incidence of HFS was 64.86%,the incidence of diarrhea was 37.84%,and the incidence of HFS and diarrhea was 29.73%.21 bile acids were detected in 91 tubes of plasma samples collected from 25 patients with colorectal cancer.The results of t test showed that the plasma concentrations of 12-KLCA,GDCA and GHCA in patients with colorectal cancer were associated with capecitabine induced nausea;the concentrations of GLCA and 23-NCA were associated with hand foot syndrome;the concentrations of TCA,GCA,7-KLCA,GUDCA,GCDCA,GHCA and GDCA were associated with leucopenia;the concentrations of TCA,GCA,7-KLCA,12-KLCA,GCDCA,GHCA and GUDCA were associated with granulocytopenia;and the concentrations of TCA,GCA,GHCA and GCDCA were associated with thrombocytopenia.No bile acids associated with vomiting,diarrhea and anemia were found.A total of 25 bile acids were detected in mouse plasma,through the comprehensive analysis of orthogonal partial least squares discriminant analysis,variable projection importance analysis and statistical test,it is found that the concentrations of 23-NCA,Iso-LCA and Iso-DCA in the baseline blood samples of diarrhea mice and non-diarrhea mice were different.There were significant differences in the plasma concentrations of Apo-CA,Iso-DCA and 7-KDCA between diarrhea mice and non-diarrhea mice;For hand-foot syndrome,no different bile acids were found.Conclusion:There is a correlation between the levels of bile acids in the plasma of patients with colorectal cancer and various adverse reactions caused by capecitabine,suggesting that bile acid could be a potential biomarker system for capecitabine adverse reactions.Animal experiments show that the plasma concentrations of 23-NCA,Iso-LCA and Iso-DCA in mice may be used as early warning biomarkers for diarrhea induced by capecitabine,while Apo-CA,Iso-DCA and 7-KDCA may serve as biomarkers for the diagnosis and grading of diarrhea.Among them,the correlation between Iso-DCA and capecitabine induced diarrhea is the most close,which may be used as an early warning biomarker for diarrhea,as well as a potential biomarker for the diagnosis and grading of diarrhea,which should be paid more attention to in subsequent studies.