| BackgroundChina has a high incidence of liver disease, which represents a serious health care problem and a high burden of national economy. Thus, the early diagnosis of liver disease and further investigation of the pathogenesis of chronic liver disease are important.Liver is an important metabolic organ of body. It plays a pivotal role in the metabolism process. If the liver is injured, it will inevitably lead to the corresponding changes of metabolic networks. Metabonomics is considered to represent the level of detection of the disease from the whole state of metabolite changes, it therefore plays a important role in the early diagnosis and mechanism study of liver disease. Fecal metabolites are considered the product of the microbial-mammalian co-metabolism. Metabolite compositions and variations of feces reflect the status of the gut microbiome community but also bridge the relationships between symbiotic microbes and the host's (human) health. Therefore, application of fecal supernatant metabonomics method to study the different stages of liver disease is important for understanding the relationships between the alteration of intestinal microflora and hepatic damage.The symbiotic gut microbiome exerts a strong influence on the metabolic phenotype of the mammalian host and participates in extensive microbial-mammalian co-metabolism. Liver and intestinal microflora exist a close relationship of anatomy and physiological functions through intestine-liver axis. Previous studies demonstrated that there was intestinal flora imbalance in liver disease. Yet the relationship between the alteration of gut microflora and liver disease has not been addressed.In the present study, we tried to establish the extraction and ultra performance liquid chromatography coupled with mass spectrometry (UPLC/MS) metabonomic technologies to investigate metabolic variations of the feces samples. Then metabolic variations in patients with acute hepatitis, chronic hepatitis, liver cirrhosis, liver cancer were investigated using this metabonomic method. Meanwhile, We observed the changes of six major kinds of fecal bacteria in patients with liver cirrhosis using the real-time PCR with SYBR Green 1. We further analyzed the effect of changed intestinal microflora on the metabolism in patients with liver cirrhosis. Part I Establishment of fecal metabonomic technologies based on UPLC/MS and application of this method on the study of different liver diseasesMethods:Our study focused on the ratio of methanol/stool (v/g), the size of filter membrane, and the electrospray ionization mode on the extraction of fecal metabolites. Metabolic variations in patients with acute hepatitis, chronic hepatitis, liver cirrhosis, liver cancer using this metabonomic technologies were investigated. Combined with multivariate data analysis, we explored the dynamic changes law of the fecal metabolic spectrum in patients with different liver disease, and assessed potential of fecal metabolic profile in the diagnosis of different stages of liver disease.Results:We established fecal metabonomic technologies based on UPLC/MS. The ratio of methanol/stool (v/g) was 600μl/0.2g, the size of filter membrane was 0.22μm, and positive electrospray ionization mode was choosed. This method validations had a high sensitivity, high resolution and a good repeatability. It applied to detect fecal metabolites rapidly. We investigated metabolic variations in patients with acute hepatitis, chronic hepatitis, liver cirrhosis, liver cancer using this metabonomic technologies based on UPLC/MS. Scores scatter plot of the principal components analysis (PCA) showed significant difference in endogenous metabolite profiles was observed in the healthy controls and patients with different liver disease. By using OPLS-DA, healthy controls and acute hepatitis, chronic hepatitis, liver cirrhosis, or hepatocellular carcinoma were distinguished, which indicated this model could be regarded as a diagnostic tool. We also indentified three major groups of metabolites, The indentified metabolites were as following:a strong increase in lysophosphatidylcholines, a dramatic decrease in bile acids and bile pigments. We considered that fecal bile acid levels might be associated with liver disease progression. Part II Effects of gut microflora on metabolic profiles in patients with liver cirrhosisMethods:After extraction from the feces of total DNA, using real-time PCR with SYBR Green 1 for quantifying the case of Lactobacillus, Bifidobacterium, Bacteroides, Enterococci, Enterobacter, Clostridium.,We analyze the UPLC/MS data using Mann-Whitney Test, Combined with multivariate data analysis to find potential biomarker in patients with liver cirrhosis.Results:We observed the changes of six major kinds of intestinal bacteria in patients with liver cirrhosis by using the real-time PCR with SYBR Green 1.Compared with healthy controls, the number of Bacteroides genus, Clostridium and Bifidobacterium were decreased significantly (P＜0.05), on the contrary, the number of Enterobacter was increased significantly (P＜0.01). By using PCA or PLS-DA, the patients with liver cirrhosis could be distinguished from health controls. Using unequal variance t-tests,2393 peaks were observed with significant difference, approximately 74.0% of which were due to decreased fecal metabolite concentrations in liver cirrhosis vs healthy controls. Integrating multivariate data analysis, we indentified six major groups of metabolites, the indentified metabolites were as following:a significantly increase in lysophosphatidylcholines, aromatic amino acids and fatty acids, a dramatic decrease in bile acids and bile pigments an elevated acylcarnitines.ConclusionsWe established a fecal metabonomic technologies based on UPLC/MS, and investigated metabolic variations in patients with acute hepatitis, chronic hepatitis, liver cirrhosis, liver cancer using this metabonomic technologies. Our results suggest that such a metabonomics method might play an important role in digging the great potential of fecal metabolic profile in different liver disease diagnosis. Our results confirmed that the existence of an imbalance of intestinal flora in the patients with liver cirrhosis by using the real-time PCR with SYBR Green 1, which might have a diverse effect on the fecal metabolic profiles, especially on the bile acid and bile pigment.
|
PDF Full Text Request