Metabolic Profiling Analysis Of Different HBV DNA Levels In CHB Patients Based On UPLC-MS

Objective: To study the differences of small molecular compounds in chronic hepatitis B patients with different levels HBV DNA,based on ultra performance liquid chromatography-mass spectrometry platform,then provide new serum markers for the clinical diagnosis,efficacy evaluation,prognosis and long term follow up of chronic hepatitis B and further explore whether these differences related to the pathogenesis of HBV.Method: All subjects were according with the diagnostic criteria of the Chronic Hepatitis B Prevention and Treatment Guidelines(2015)made by Chinese Medical Association of Infectious Diseases and Hepatology,and they were also met the other inclusion and exclusion criteria of this study.We based on the ultra performance liquid chromatography-mass spectrometry system platform to detect the metabolism compounds of people with chronic hepatitis B,25 cases positive HBV DNA and 10 cases negative HBV DNA patients included.Raw data performed by SIEVE V2.1 software.And noise filtering,resolution of overlapping peaks,peak alignment,peak matching,standardization and normalization were performed.Positive and negative peak table,were saved as Excel table.According with 80% rule,missing values were excluded.Excel data was import into SIMCA-P 13.0 software,and a partial least squares-discriminant analysis(PLS-DA)method was applied to perform pattern recognition analysis.Calculating variable importance in projection of each variable in partial least squares model,and combining with score plots,loading plots and nonparametric test,we found the potential biomarkers.Finally,we depended on m/z and retention time searching on databases to confirm the substance.SPSS19.0 software was used for statistical analysis.Result: Basing on two independent samples t-test,showed that there was no statistically significant in age distribution between positive and negative HBV DNA group(P = 0.942,39.64 ± 11.09,43.60 ± 11.49).And basing on Fisher,s Exact Test,the sex distribution of the positive and negative HBV DNA group was similar(P=0.258).Positive HBV DNA group achieved higher alanine aminotransferase,aspartate aminotransferase and ?-glutamyl transferase levels than negative HBV DNA group(P=0.010,P=0.003 and P=0.009).And the serum levels of albumin,globulin,total bilirubin,alkaline phosphatase and cholinesterase between the positive and negative HBV DNA group were similar.By ultra performance liquid chromatography-mass spectrometry system,336 kinds of small molecular compounds were identified.In the negative ion mode,we identified 151 kinds of compounds,and in the positive ion mode we identified 185 kinds of compounds.We successfully constructed metabolic profiling analysis model of positive HBV DNA and negative HBV DNA by partial least squares((R2= 0.00,0.666),Q2 =(0.00,-0.201)).Finally,two kinds of potential biomarkers were found.In the negative ion mode,we screened out isocitrate,which in HBV DNA negative group was significantly higher than that in HBV DNA positive group;in the positive ion we screened out lysophosphatidylethanolamine(22: 4),which in HBV DNA positive group was significantly higher than that in HBV DNA negative group.Conlusion: 1.Ultra performance liquid chromatography-mass spectrometry system and PLS-DA model can be used as new method of HBV DNA level assessment.2.Based on metabolomic method,isocitrate and lysophosphatidylethanolamine(22: 4)are found can be used as potential biomarkers to distinguish positive and negative HBV DNA and can show different pathophysiological states of body.3.Krebs cycle pathway and phospholipid metabolic pathway may provide a new perspective and vision for the research of hepatitis B pathogenesis.