| [Background and Objectives]The purpose of this study is to use Triple quaternary bar mass spectrometry for liquid chromatography in exploring the metabolic profile of the hepatitis B virus associated liver diseases patients,like chronic hepatitis b,liver cirrhosis,hepatocellular carcinoma and hepatic,and establish the metabonomics diagnosis model between the four groups.At the same time,metabolic substances(amino acids and carnitine)related to the different disease stages were screened out to provide a new method for clinical disease stage definition and prognosis evaluation.[Methods]96 patients hospitalized in jinan infectious disease hospital from January 2018 to December 2018 were randomly selected,including 24 patients with chronic hepatitis b,31 patients with Hepatitis b cirrhosis,29 patients with primary liver cancer,12 patients with liver failure(6 improved,6 died),in addition,And the medical examination of 25 cases of our hospital staff as a normal control group.Blood was taken from the tip of the hollow finger in the morning on the dry blood filter,natural drying at room temperature and stored at 4 ? refrigerator.Set aside.The samples were prepared by extraction,reaction(derivatization)and resolubility,and the contents of various amino acids and acylcarnitine were determined by tandem four-step mass spectrometry.Statistical analysis:R language 3.5.3 "ropls" package was used for analysis,the score map was drawn using unsupervised principal component analysis(PCA),and then 70%of patients in each group were randomly selected to form the training set for the construction of the opls-da model.The four dichotomized opls-da models were:distinguish between the normal control group and the chronic hepatitis b group,the chronic hepatitis b group and the liver cirrhosis group,the liver cirrhosis group and the hepatocellular carcinoma group,as well as the liver failure improvement group and the death group,and the remaining 30%of patients were used to test the accuracy of the training model.By using the variable projection importance value(VIP)in the binary classification opls-da model,VIP>1 was used as the screening standard to screen out the metabolites with differential significance between each group,and combined with univariate statistical analysis(mann-whitney U test)to screen the metabolites with difference,P<0.05 was considered statistically significant SPSS23.0 software was used to analyze and process the data.M(P25,P75)was used to express the measurement skewness distribution data.Kruskal-wallis H test was applied to nonparametric test and pairwise comparison of multiple independent samples.The chi-square test of multiple independent sample contingency tables was applied to the classification data.Test level =0.05,P<0.05 was statistically significant.[Results]1.Results of principal component analysis(PCA):The normal control group was mainly distributed in the first and second quadrants;Chronic hepatitis b group mainly distributed in the second quadrant;Hepatitis b cirrhosis group mainly distributed in three,four quadrants;Primary hepatocellular carcinoma group mainly distributed in the third and fourth boundaries;The improvement group of liver failure mainly distributed in the third quadrant,and the death group mainly distributed in the first quadrant.It can be seen that the principal component analysis(PCA)method is not enough to distinguish the above groups and build the opls-da model for them.2.The orthogonal partial least-squares discriminant analysis(OPLA-DA)results:In order to the four groups,liver cirrhosis group and primary liver cancer group,and hepatic failure improvement group and death group classification of the four OPLS-DA model fitting of R2Y were 0.89,0.802,0.884 and 0.998;The predictive power Q2Y of the model is 0.721,0.327,0.482 and 0.884,respectively.The prediction accuracy of the model was 0.867,0.647,0.944 and 0.5,respectively.3.Comparison of amino acid levels between different groups of HBV-associated chronic liver diseases:(1)The comparation of the normal control group and the chronic hepatitis b group:the levels of Valine(Val),Proline(Pro)and ornithine(Orn)are decreased,while tyrosine(Tyr)is increased in the chronic hepatitis b group.(2)The comparation of the chronic hepatitis b group and the hepatitis b cirrhosis group:the hepatitis b cirrhosis group showed decreased branched-chain amino acid(leucine,isoleucine and valine)and proline level,while increased citrate level.(3)The comparation of the hepatitis b cirrhosis group and primary liver cancer group:the level of Pro(proline)in primary liver cancer group was significantly increased.(4)The comparation of the improvement group and the death group in liver faliure:the level of arginine significantly increased in improvement group.4.Comparison of acylcarnitine levels among different hbv-associated chronic liver disease groups:(1)The comparation of the normal control group and the chronic hepatitis b group:the level of C4OH and C3DC,C5DC and C6OH,C8:1,C8,C10:2,C10:1,C14OH,C18,C18:1 OH and C3/C2 are decreased in chronic hepatitis b group,so the levels of acylcarnitine in short and medium and long chains were decreased.(2)The comparation of the chronic hepatitis b group and the liver cirrhosis group:the level of C5OH + C4DC is decreased,while the level of C5DC + C6OH,C16,C18:2,C18:1 and C18 are increased in the liver cirrhosis group,so the levels of short chain acylcarnitine were decreased,while the medium and long-chain acylcarnitine were increased.(3)No difference in acylcarnitine was found between HBV cirrhosis and HCC.(4)The comparation of the improvement group and the death group in liver faliure group:the level of C3,C16:1 and C160H are decreased in the death group of liver faliure group,so the levels of acylcarnitine in short and long chains were increased.[Conclusions]1.The opls-da model was established to find the method to distinguish the difference of amino acids and acylcarnitine between the hbv-associated chronic hepatitis b,hepatitis b cirrhosis,primary liver cancer,liver failure improvement group and death group.2.After the analysis of the detected differences in amino acids and acylcarnitine,we found that the detection of the changes in serum amino acids and acylcarnitine levels by liquid phase tandem mass spectrometry may be a more effective method for monitoring the progress and prognosis of hbv-associated chronic liver disease.3.By the comparation of amino acids and acyl carnitine levels among chronic hepatitis b,hepatitis b virus related liver cirrhosis and primary liver cancer,liver failure improvement group and death group,it is concluded that when the hepatitis b virus related chronic liver diseases occur,the branched chain amino acid and aromatic amino acid metabolism,the synthesis of the non-essential amino acid and the urea cycle are affected,and as the degree of hbv-associated chronic liver disease is aggravating,the metabolic pathway damage also becomes more serious.About the acylcarnitine levels:compared with the normal control group,chronic hepatitis b group showed a decrease in short,medium and long chain acylcarnitine.Compared with chronic hepatitis b group,the level of short acylcarnitine in cirrhosis group decreased obviously,while that of medium and long acylcarnitine increased obviously.Compared with the death group,the shorter and longer acylcarnitine was significantly increased in the liver failure improvement group.No difference in carnitine levels was found between the hepatitis b cirrhosis group and the primary liver cancer group.4.The levels of C3,c16:1 and C160H in the improved group of liver failure were significantly increased,which may have predictive value for the prognosis of liver failure.Therefore,it is necessary to increase the sample size and deepen the understanding. |
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