Construction Of Amino Acid Profile And Its Application In The Study Of Hyperlipidemia

Backround:Amino acids are basic unit that construct material basis of life.Under normal physiological conditions,amino acid profile is in an equilibrium state in vivo,imbalance of amino acid profiles is the cause or manifestation of many diseases.Studies have shown that amino acid profiles are increasingly used in the field of biomedical research such as health assessment,disease diagnosis,efficacy evaluation,etc.Amino acid profile is one of the important technical toos in studying major metabolic diseases and their prevention and control strategies.Professor Guo first proposed the innovation theory of "Glucolipid Metabolic Disorders(GLMD)",it is considered that GLMD is characterized by disorders of glucolipid metabolism and involves five core pathogenesis,including neuroendocrine disorder,insulin resistance,oxidative stress,inflammation and alteration of intestinal flora.However,the dynamic changes of amino acid profile of GLMD are not clear.Hyperlipidemia is the most common clinical manifestation of glycolipid metabolism,how to effectively prevent and control hyperlipidemia is a major and arduous task.Tianhuang Pian(THP),a representative prescription of Professor Guo summaried according to long-term clinical experience,has significant lipid-lowering effect on hyperlipidemia.Previous studies have shown that THP is involved in regulation of oxidative stress,inflammation,amino acid metabolism and energy metabolism.However,there is little report on characterization of hyperlipidemia based on amino acid profile and intervention mechanism of THP.Objective:To establish qualitative and quantitative analysis method of plasma amino acids,construct amino acid profile of hyperlipidemia and study effect and mechanism of THP intervention in hyperlipidemia based on amino acid profile.Methods:1.Study on fingerprint of amino acids in plasma: comprehensive qualitative analysis of plasma amino acids were performed using UPLC-Q/TOF MS technology,including direct qualitative and derivatization qualitative.After AQC derivatization for plasma amino acids,chromatographic mass spectrometry conditions were optimized and methodological investigation was carried out to establish a quantitative analysis method of plasma amino acid AQC pre-column derivatization combined with UPLC-QDa.Liquid chromatography and mass spectrometry conditions were optimized,then method validation were performed to establish AQC pre-column derivatization combined with UPLC-QDa quantitative analysis method for plasma amino acids.2.Construction of hyperlipidemic amino acid profile: hyperlipidemic rat model was established by high-fat emulsion gavage,20% high fructose free diet and high-fat diet.Biochemical analyzer was used to analysis the concentration of blood glucose and lipid for model evaluation,and quantitative analysis of amino acids in plasma of hyperlipidemia was performed by established method.Combined with multivariate statistical analysis,amino acid biomarkers in plasma of hyperlipidemia were identified,then amino acid profile of hyperlipidemia was constructed.Network pharmacology was used to analyze targets and signaling pathways of characteristic amino acids,explaining the biological significance of characteristic amino acids.3.Intervention of THP in hyperlipidemia: taking plasma of hyperlipidemic mouse as the research subject,quantitative analysis of amino acids was performed based on AQC pre-column derivatization combined with UPLC-QDa analysis technology.Combined with multivariate statistical analysis,amino acid biomarkers were identified,then amino acid profile of hyperlipidemia was constructed and intervention of THP on characteristic amino acids was analyzed.Results:1.Study on fingerprint of amino acids in plasmaCombined with direct and derivative analysis methods,a total of 52 amino acids in plasma were qualified.36 amino acids were simultaneously quantified in 10.7 min by AQC pre-column derivatization combined with UPLC-QDa analysis technology.2.Construction of hyperlipidemic amino acid profileHyperlipidemic rat model was established by high-fat emulsion gavage,20% high fructose free diet and high-fat diet.Metabolic disorders occurred in blood glucose,lipid and amino acids.Plasma amino acid profile can effectively distinguish normal and hyperlipidemia group.The constructed amino acid profile of hyperlipidemia has a total of 20 amino acids,including five common amino acids,namely Glu,Sar,?-AIB,Cys2 and Pip.The trend is that both Glu and Sar are decreased,?-AIB,Pip and Cys2 are elevated or decreased.The above-mentioned characteristic amino acids of hyperlipidemia have three common targets,namely PTGS1,PYGM and PTGS2,it is predicted that they participate in regulation of lipolysis in adipocytes,starch and sucrose metabolism,insulin resistance,etc signaling pathways.3.Intervention of THP in hyperlipidemiaThe plasma amino acid profile of hyperlipidemic mice has a total of 13 amino acids,including Pip,Ser,Ala,Met,Aad,?-AIB,Car and Asn,the trend is that levels of Pip,Ser,Ala,Met,Aad,?-AIB,Car and Asn are elevated,while levels of Sar,Phe,Ile,Orn and Val are decreased.In terms of drug intervention,THP has reversal effect on plasma Sar,Aad,?-AIB and Car.Compared with atorvastatin,THP can uniquely regulate Sar,acting on PPIG and GNMT targets of hyperlipidemia and participate in glycine,serine and threonine metabolic pathways.Conclusion:In this study,52 amino acids in plasma were identified,AQC pre-column combined with UPLC-QDa method for simultaneous quantification of 36 amino acids in 10.7 min was established.Balance of amino acid metabolism in hyperlipidemia established by high-fat emulsion gavage,20% fructose diet and high-fat diet is disturbed,amino acid profile can be used to characterize hyperlipidemia.In terms of intervention,amino acid profiles also can be used to characterize metabolic changes in vivo after THP administration.Compared with atorvastatin,THP can uniquely regulate Sar,acting on PPIG and GNMT targets of hyperlipidemia and participate in glycine,serine and threonine metabolic pathways.