| Objective:At present, metabolomics approach has been widely used in serum, plasma, tissue, and a variety of physiological fluids,many of which are ascites, urine and other samples. In these samples, urine not only contains a large number of metabolites, excreted from the body but also non-invasive access to patients and become a hot research sample by scientists.The study is intended to establish the non-invasive urine metabolomics experiments based on HPLC/MS technology and make a comprehensive analysis of hepatocellular carcinoma (HCC) on metabolic fingerprinting, guiding diagnosis and treatment of disease.We applied this theory, combining with modern biological mass spectrometry, analysis the metabolic spectrum difference among hepatocellular carcinoma, liver cirrhosis patients and normal control subjects in the overall level and carry out the metabolomics study on liver cancer caused by hepatitis B and liver cirrhosis patients, exploring the new ideas for disease mechanism.Methods:(1) The pretreatment method for biofluid was established successfully. Proteins make the largest affect in biological samples (This article refers to urine).If we detect protein by chromatography, protein will deposite and clog the column This test established a pretreatment method by screening.Urine was centrifuged at 15000rpm to separate protein, and add urine supernatant with 0.1% acetonitrile according to the ratio of 1:4(v/v)mix and vortex. Pipet resuspension and degas it through filter membrane of 0.45 um.Then place the samples on the HPLC/MS. directly.(2)We established High performance liquid chromatography/mass spectrometry (HPLC/MS)-based on metabolomics platform of HCC urine and apply the feasibility of the application of HPLC/MS technology in the early diagnosis of HCC, Through the urine of patients with HCC metabolites,we guess liver cancer-related changes to infer the metabolic changes in the channel,and serach the change discipline associated stability of the metabolites.We use HPLC/MS technology to make a differential diagnosis among HCC,cirrhosis and normal control group of metabolic spectrum.Results:(1) The steps of pretreatment is relatively simple to use organic solvent processing urine,which does not affect the analysis results,and have sufficient sensitivity,the contamination instrument system is also small, so it may as a group of routine urine dilution method on urine metabolic study.(2)We initial haved established HPLC/MS based metabonomics preliminary study of HCC of urine,by comparing the urine metabolite profiles between HCC caused by hepatitis B virus and normal group,through database identification,we get llendogenous metabolites.Tyrosine, glutamate and so on were significantly higher in the patient group,Glycine, leucine, and others decrease notably,there are significant changes in some of lipids;between the metabolite profiles of HCC and cirrhosis, we get 6 endogenous metabolites,most of which are leukotrienes and urobilinogen. With metabolomics data,we applicate OPLS-DA and other discriminant analysis to find that three groups of patients were able to complete clustering, so we can use metabolomics technology to make differential diagnosis among three groups.Conclusion:By the study of urine metabolomics among normal control, cirrhosis and HCC, we find metabonomics data has a good diagnosis of the disease, but also has a role in the efficacy assessment.,which can reveal some of the pathological mechanism of development of HCC. As a new branch of modern life science, metabolomics s has important clinical value on the study of the pathogenesis and the diagnotis of liver disease... |
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