High Performance Liquid Chromatography Of Aromatic Amino Acids In Serum And Its Clinical Application Are Discussed

Objective: The aim of this study was to establish a simple and rapid method for the determination of aromatic amino acids in serum by reversed phase high performance liquid chromatography with ultraviolet detection (HPLC-UV),and explore the clinical significance of serum aromatic amino acids determined in patients with hepatitis, hepatocirrhosis or schizophrenia.Method: The chromatographic conditions used were a reversed-phase Nova-pak C18 column (4 μ m,3.9mm id× 150mm, Waters) at room temperature and a flow rate of 1.0mL/min with detection wavelength at 215nm and acetonitrile-water(6:94 in volume ratio, pH=3.4) as mobile phase. Serum samples was precipitated by 5% perchloric acid solution and centrifugalized to move protein and then assayed by HPLC-UV within 10min. Effects of pH value of the mobile phase, the fraction of organic phase, protein precipitants and detection wavelenghth on the response and retention were explored. Interferences of other amino acids and dopamines were also studied. Subjects studied were divided into 4 groups: normal control group(86 cases), hepatitis group(35 cases), hepatocirrhosis group(35 cases), and schizophrenia group(32 positive cases, 26 negative cases).Results: The intra-day precisions(CV) of tyrosine, phenylalanineand tryptophan were 1.79%,2.57%,1.41% respectively; the inter-day precisions(CV) were 2.66%,3.99%,2.46% respectively; the linear range were 5.5-550.0 μ mol/L,6.1-610.0 μ mol/L,4.9-490.0 μ mol/L; the detection limits were 0.6> 0.5,0.4 μ mol/L; the average recovery rates were 99.0%, 99.3%, 98.0% respectively. The interferences of Ala, Val, He, Leu, Met and 5-hydroxytryptamine, 5-hydroxyindole acete acid, dopamines were never found with the chromatographic conditions used in this trial.The concentrations of Tyr, Phe and Trp in Normal male adults were (43.59±6.68) μ mol/L, 62.52 ±7.60) μ mol/L, (44.41±7.90) μ mol/L, and female adults were (40.05±7.96) μ mol/L, (57.57± 10.10) μ mol/L, (38.90 ± 5.25) μ mol/L respectively. The levels of serum aromatic amino acids were higher in hepatitis and hepatocirrhosis groups than those in normal control group(p<0.01). There was a positive correlation between Tyr/Phe and ALT in hepatocirrhosis. There were no significant changes of the values of aromatic amino acids, Trp/Tyr, Trp/(Tyr+Phe) between normal control and negative Sch group(p>0.05). Compared with normal control and negative Sch group the levels of Phe, Tyr in positive Sch group were significantly higher(p<0.05), the values of Trp, Trp/Tyr, Trp/(Tyr+Phe) were significantly lower(p<0.05).Conclusion: HPLC established in this trial for determination of serum aromatic amino acids was simple, rapid, sensitive and accurate,and convenient for clinical and scientific study. The concentrations of aromatic amino acids in hepatitis and hepatocirrhosis group increased significantly, and the value of Tyr/Phe could reflect liver function. The values of serum Trp/Tyr and Trp/(Tyr+Phe) in schizophrenia could reflect the metabolism of amino acids.