| Part 1.A sensitive and accurate method to measure amino acids in maintenance hemodialysis patients by using LC-MS/MSPurpose: To establish an economical,rapid,accurate,reliable,and sensitive method for simultaneously measure amino acids in patients by liquid chromatography-tandem mass spectrometry(LC-MS/MS).This validated method was further used to explore the clinical significance of various free amino acid levels in the plasma of maintenance hemodialysis patients.Methods: We randomly collected the blood of 80 healthy volunteers and prepared the samples to be tested for LC-MS/MS analysis.The stable isotope-labeled amino acid was used as the internal standard,and the standard curve was draw.By using LC-MS/MS,we can simultaneously quickly,sensitively and accurately determine the levels of various free amino acids in plasma.Results: The levels of various free amino acids in normal human plasma are: glycine 9.31-25.22 μg/ml,proline 20.04-61.72 μg/ml,lysine 22.95-50.29 μg/ml,leucine 10.49-27.28 μg/ml,Tryptophan 8.99-23.08 μg/ml,Valine 20.62-51.19 μg/ml,Isoleucine 8.53-30.56 μg/ml,Ornithine 5.40-16.52 μg/ml,Histidine 10.97-22.04 μg/ml,Threonine 3.07-29.43 μg/ml,Arginine 5.05-15.85 μg/ml,Methionine 2.98-6.91 μg/ml,Serine 10.51-28.05 μg/ml,Phenylalanine 7.88-16.02 μg /ml,Taurine 2.38-7.94 μg/ml,Cysteine 4.81-15.34 μg/ml,Tyrosine 13.59-32.80 μg/ml,Alanine 31.36-205.61 μg/ml,Asparagine 1.66-15.25 μg/ml,Aspartate 0.75-11.41 μg/ml,Glutamate 5.89-22.95 μg/ml.The reportable range of various amino acids is large,and their detection limits,accuracy,precision,and matrix effects are all acceptable.Conclusion: LC-MS/MS method was successfully established to determine the levels of 21 free amino acids in patients’ plasma at the same time,and can be applied to clinical and transformation studies.Part 2.Characteristics of amino acid profiles in maintenance hemodialysis patientsPurpose: To clarify the characteristics of the amino acid profile of maintenance hemodialysis patients,and to explore the relationship between the amino acid profile of maintenance hemodialysis patients and related clinical characteristics.Methods: A cross-sectional study was conducted to study patients undergoing maintenance hemodialysis in Shanghai Tenth People’s Hospital from January 1,2018 to March 30,2020.Baseline data such as age,gender,dialysis vintage,height,weight,renal disease,blood routine,blood biochemistry,echocardiography,and hemodialysis-related parameters were collected at the time of enrollment.The plasma was collected before and after dialysis and 21 free amino acid levels in the sample were detected by LC-MS/MS on the day of enrollment.Univariate and multivariate analysis were used to analyze the characteristics of various free amino acids in maintenance hemodialysis patients,and to evaluate the correlation between different amino acid levels and clinical characteristics.Results: 1)Compared with normal volunteers,maintenance hemodialysis patients had higher levels of leucine,valine,histidine,tyrosine,tryptophan,lysine,serine,taurine,alanine,Aspartate levels(P<0.05);maintenance hemodialysis patients had lower levels of isoleucine,phenylalanine,glycine,ornithine,arginine,cysteine,and glutamate(P < 0.05);there was no statistical difference in the levels of methionine,proline,threonine and asparagine between the two groups(P > 0.05).2)The amino acid profiles of maintenance hemodialysis patients was correlated with dialysis adequacy,lipid metabolism,glucose metabolism,and inflammatory status.3)The plasma concentrations of 20 amino acids were all decreased in maintenance hemodialysis patients,except that the concentration of aspartic acid increased after a single dialysis session.4)There was no difference in the effect of a single session of high-flux hemodialysis versus low-flux hemodialysis on plasma free amino acid levels.5)After adjusting for factors such as patient age,dialysis vintage,BMI,albumin,dialysis mode,renal disease,the concentrations of leucine,isoleucine,histidine,phenylalanine,lysine,ornithine,arginine,asparagine and glutamate in the male group were higher than those in the female group(P < 0.05).Conclusion: The amino acid metabolism of maintenance hemodialysis patients was seriously disordered,and the levels of various amino acids were higher than those of normal people,especially the level of branched chain amino acids,which was significantly different from that of healthy control group.The amino acid profile of maintenance hemodialysis patients is related to dialysis adequacy,lipid metabolism,glucose metabolism,inflammatory state and so on.In maintenance hemodialysis patients,except aspartic acid,the levels of other 20 plasma free amino acids decreased significantly after a single dialysis session;There was no difference between single high flux hemodialysis and low flux hemodialysis on the level of plasma free amino acids.Amino acid profiles in maintenance hemodialysis patients were correlated with gender.Part 3.Clinical significance of branched-chain amino acids on clinical prognosis of maintenance hemodialysis patientsPurpose: To investigate the effect of branched chain amino acids on the clinical prognosis of maintenance hemodialysis patients.Methods: A retrospective cohort study was conducted on the patients with maintenance hemodialysis in Shanghai Tenth People’s Hospital from January 1,2018 to March 30,2020.The baseline data of patients were collected retrospectively,such as age,gender,dialysis vintage,body mass index,renal disease,dialysis related parameters,blood routine,blood biochemistry,plasma branched chain amino acid concentration(including leucine,isoleucine and valine),dialysis adequacy,cardiac ultrasound.Patients were divided into high concentration group and low concentration group according to the baseline concentration of branched chain amino acids,and were followed up for an average of 2 years.The primary endpoint was major cardiovascular adverse events,including nonfatal myocardial infarction,nonfatal stroke,new onset atrial fibrillation or atrial flutter,arterial embolism,hospitalization due to acute heart failure or other cardiovascular events Univariate and multivariate Cox regression analysis were used to compare the incidence of major cardiovascular adverse events between the two groups.Results: A total of 113 maintenance hemodialysis patients were included.With a median follow-up of 24 months,adjusting for gender,age,dialysis vintage,body mass index,renal disease,the analysis results showed that hemodialysis patients with high concentration of baseline branched chain amino acids had a higher risk of cardiovascular events(adjusted HR = 1.97,95% CI 1.15-3.39,P = 0.014).Hemodialysis patients in the high baseline valine concentration group had a higher risk of cardiovascular events(adjusted HR = 1.96,95% CI 1.12-3.41,P = 0.018).Conclusion: The higher level of BCAA at baseline indicates that maintenance hemodialysis patients have a higher risk of cardiovascular events in the next 2 years. |
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