| Background and objective：Hepatic encephalopathy (hepatic encephalopathy,HE) was caused by acute liverdisorder or disorders, metabolic disorder as the main features of central nervous systemdysfunction syndrome。Patients With liver disorders or disabilities (medical history,clinical manifestations and biochemical anomalies) occur neural, mental exception,such as disturbance of consciousness, behavior disorders, and coma and neurologicalsigns, after excluding other brains or mental illness, are diagnosed as hepaticencephalopathy.Minimal hepatic encephalopathy (minimalhepatic encephalopa MHE)refers to no clinical evidence of hepatic encephalopathy in cirrhotic patients can beobserved in the cognitive function, brain neurochemical/neural transmission steady,the new supersedes the old., electrophysiological parameters, blood flow to the brainand body fluid homeostasis in minor changes.Patients look like normal,but the operationcapacity and emergency response capacity reduce, can cause accidents in driving,high-altitude operations, and mechanical operation work, the prevalence is30%-80%inpatients with liver cirrhosis.More and more people attach great importance to it, butthere is currently no uniform diagnostic criteria, there is an urgent need for an objective,simple, and biochemical indicators of specific high to guide clinical diagnosis ofminimal hepatic encephalopathy.Metabolomics is a new developing discipline in recent years, not only for thequantitative description of levels of endogenous metabolites in the organism as a whole,can also be used for diagnosis of disease.An important part of study of the metabolomeis the comparative analysis of differentially expressed small molecule metabolites inhealth and disease states, biomarkers that helps people to find the early diagnosis ofvarious diseases (biological marker), and can be used for disease prognosis andtherapeutic effect evaluation.The liver is the center organs of the body responsible forenergy metabolism and material metabolism, liver function obstacle, liver metabolism disorder, the corresponding changes of metabolites, cause central nervous systemdysfunction, leading to the occurrence of hepatic encephalopathy.In this study, we use high performance liquid chromatography-mass ion trapmass spectrometry mass spectrometry (UPLC/LTQ-Orbitrap MS) technology toseparate and extract the clinical hepatic encephalopathy, minimal hepaticencephalopathy, exclusion of hepatic encephalopathy in cirrhotic and normal controlgroup in serum of patients with metabolic products, in order to find out the differencesbetween the four metabolites as the biggest sign of minimal hepatic encephalopathy thefabric.Methods:1.With permission,38patients from the Number Six Hospital of Dalian China whohad consumed hepatocirrhosis and accepted at least5years of Education were enrolledin this study.Select Group of healthy people33cases for normal control.2.Detect all of the patients with cirrhosis of liver function, blood ammonia,prothrombin time and B ultrasound,so as Child-pugh classification.3.Serum markers:High performance liquid chromatography-mass ion trap massspectrometry mass spectrometry (UPLC\/LTQ-Orbitrap MS) was detected by thetechnique of hepatic encephalopathy patients and control group, healthy controlmetabolites in serum of hepatic encephalopathy, find out the metabolite markers group.3.1sample preparation: at room temperature static thawed serum, serum400ul100ul in acetonitrile, severe concussion and30seconds, set aside for10minutes after4℃t o14000×g centrifuge for10minutes, the supernatant was lyophilized450ul freezeconcentration instrument.3.2analysis of ultra high performance liquid chromatography: ultra performanceliquid chromatography analyzer. Washing the column with10cm*2.1mm*1.7umC18column flushing using acetonitrile linear gradient, the residence time of differentsubstances in the column, in order to achieve the separation of metabolites of purpose.3.3High performance liquid chromatography-mass ion trap mass spectrometrymass spectrometry (UPLC\/LTQ-Orbitrap MS): the LTQ-Orbitrap mass analyzer,metabolites of ESI+mode operation.4.Analysis of data processing and Statistics:We use SSPS18.0statistical softwareto selected cases of liver function and blood ammonia value for non-parametertest,P>0.05showed that patients in each group of data has little difference, P<0.05shows large differences between sets of data.The Micromass Marker Lynx for each peak retention time (TR) and m/z data of each peak, ion density by standard output,principal component analysis (PCA). The non parametric test, find out the differentmetabolic product.5.Metabolites were identified by name: the precise molecular weight metabolitesname lookup.Results:1.Selected38cases of patients with hepatocirrhosis, with HE12cases, accountedfor32%; with MHE11cases, accounting for29%; This is said that the incidence ofencephalopathy is high.2..In the test, blood ammonia、liver function were detected in the three groups ofhepatocirrhosis;We find that blood ammonia monitoring is not an effective indexes ofMHE;Blood ammonia value cannot effectively distinguish MHE and HE. The degree ofinflammatory injury of liver function and synthesis function have no obvious correlationwith hepatic encephalopathy.3.Child-pugh grade is carried in the groups of hepatocirrhosis, We find nosignificant correlation between Child-pugh grade of liver cirrhosis and hepaticencephalopathy;Liver function, hepatic encephalopathy have no obvious correlation.Butin patients with hepatic encephalopathy, the severity of hepatic encephalopathy relatesto Child-pugh levels.4.The metabolite peaks spectrum and ion intensity map, PCA model and PLS-DAmodel of normal people, simple liver cirrhosis patients,HE and MHE in serum ofpatients with amine metabolite profiles find that arginase, L-tyrosine, glutamic acid,two L-phenylalanine peptide, homovanillic acid, ornithine, L-serine was graduallyincreased in normal, simple liver cirrhosis and in patients with hepatic encephalopathy,hypoxanthine and decreased gradually in normal, liver cirrhosis and hepaticencephalopathy. Compared with HE patients, changes in serum of patients with MHEand HE metabolites are mostly similar.Conclusions:1.Hepatic encephalopathy in patients with liver cirrhosis is in the high incidence,blood ammonia can not be used as the index for the diagnosis of MHE. The degree ofinflammatory injury of liver function and synthesis function and hepatic encephalopathyhad no obvious correlation.2.No correlation is found between Child-pugh grade of liver cirrhosis and hepaticencephalopathy.But in patients with hepatic encephalopathy, the severity of hepatic encephalopathy relates to Child-pugh levels.3.Two hydrogen thymine, arginase, L-tyrosine, glutamic acid, two L-phenylalanine peptide, homovanillic acid, ornithine, L-serine, hypoxanthine can beused as a mild hepatic encephalopathy metabolic markers group. Compared with HEpatients, changes in serum of patients with MHE metabolites and HE most similar. |
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