| Non-alcoholic fatty liver disease(NAFLD)has rapidly become the most common cause of chronic liver disease worldwide.NAFLD can develop into liver fibrosis,liver cirrhosis,and hepatocellular carcinoma.NAFLD refers to fatty degeneration of hepatic parenchymal cells caused by excessive alcohol intake,autoimmunity and virus infection.Although the mechanism of NAFLD and the progress mechanism of NASH are still unclear,it has been suggested that the disorder of branched-chain amino acids may be related to the same forms of liver injury.In chronic liver diseases,especially cirrhosis,branched chain amino acids decrease and aromatic amino acids increase.Quantitative determination of the concentration of various substances in the brain plays an important role in the control of clinical diseases.At present,the simultaneous determination of amino acids and monoamines by HPLC-FLD is seldom used.Therefore,this paper is divided into three parts to discuss the simultaneous determination of 13 substances by HPLC-FLD and the changes of 13 substances in the course of a non-alcoholic fatty disease.Part Ⅰ:Establishment of a method for the determination of 13 amino acids in mouse brain tissue.Objective:To establish an HPLC-FLD method for simultaneous determination of 13 substances in mouse brain tissue.Method:Agilent 1260 high-performance liquid chromatography was used in the experiment.The excitation and emission wavelengths were 340 and 455 nm,respectively.ODS2 column used and the temperature set at 30℃.The gradient elution method of high-performance liquid chromatography was used to determine the concentration at a flow rate of 1.0 mL min-1.The mobile phase A is acetonitrile.The mobile phase B is 30 mmol.L-1 sodium acetate.All mobile phases were filtered and degassed by ultrasound for 10 minutes.The injection volume was 20 μL and the total running time of the sample was 45 minutes.Results:Asp,Glu,Gln,Gly,Tau,Tyr,GABA,Trp,Met,Val,Phe,lie,and Leu had better separation.The linear correlation coefficients R2 of the standard curves of 13 target substances are all greater than 0.9996.Except for Val and Phe(0.9 μmol·L-1),LOQs of most compounds were lower than 0.1μmol·L-1,and all LODs were lower than 0.05μmol·L-1.At low,medium,and high concentrations,the inter-day precision were less than 2.23%,4.41%,and 2.08%;the intra-day precision were less than 8.65%,3.90%,and 4.88%respectively.The accuracy expressed as relative recovery(RR)ranged from 77.52%to 111.58%.The recovery of extraction ranged from 75.74%to 96.42%.Conclusion:The established method of HPLC-FLD for the detection of 13 analytes is suitable for the separation and determination of complications related substances in NAFLD.Part Ⅱ Determination of 13 Amino Acids in Brain Tissues of Male Mice of C57BL/6,RIP3,Mfge8,and C3R StrainsObjective:To determine the contents of 13 amino acids in brain tissues of male mice of 4 strains C57BL/6,RIP3,Mfge8,and C3R,and study the relationship between the changes of 13 amino acids and NAFLD.Method:The method established in the first part of this paper was used.Results:Most of the amino acids in the brain tissue of C57BL/6 mice fed with MCD feed changed after 9 weeks.With the increase of feeding time,these amino acids changed differently.Asp,Gln,GABA,and Ile contents could return to normal to a certain extent,but the amino acid content would decrease significantly after 18 weeks of feeding.By comparing RIP3,Mfge8,and C3R,it was found that there was no significant difference in the content of amino acids in the brain of different strains of mice at the same time when they fed with MCD feed for 9 weeks,and the changes of Asp in the brain tissues of the three strains were consistent.When the MCD diet fed for 18 weeks,the difference of amino acid content between Mfge8 and RIP3 and C3R mice was the greatest,but there was no significant difference between RIP3 and C3R mice.Conclusion:C57BL/6 and Mfge8 are suitable for the establishment of non-alcoholic lipopathy model mice. |
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