| Objective:1.To establish a simple and rapid method for simultaneous determination the concentrations of serum PHE, TYR and TRP in healthy persons by high performance liquid chromatography with fluorescence detection.2.To explore the clinical significances of serum PHE, TYR and TRP in patients with Chronic renal insufficiency(CRI). Methods:1.Serum samples were deproteinized with 5%(v/v) perchloric acid at equal volumn and injected 20μl supernatant to analysis directly. Separation was achieved on the Megres C18 column (4.6mm×250mm i.d.,5μm, Hanbon Co., Ltd. Jiangsu,China) with a mobile phase consisting of 10%acetonitrile in water(V/V).The flow rate was set at 1.OmL/min. The eluted AAA was scanned by the programmed wavelength detection setting.The effects of various aspects on operation and determination were examined to establish optical assay conditions, such as detection wavelength, PH value, the content of organic solvent in the mobile phase and flow rate. The analysis of PHE, TYR and TRP by retention time and superposition method. External standard method as the quantitative method.The influences of common substances which produce natural fluorescence were investigated.2.The concentrations of serum PHE, TYR and TRP in healthy persons (n=100) were analyzed by HPLC-FLD, and the total of AAA and the ratio of TYR to PHE were calculated.3. The concentrations of serum PHE, TYR and TRP in patients with CRI (n=80) were determined by HPLC-FLD and the total concentration of AAA and the ratio of TYR to PHE were calculated. ROC was used to evaluate its sensitivity, specificity and methodology efficiency for CRI.To analysis the correlation between the serum AAA, TYR, PHE and TRP concentrations) and the other biochemical indicators, and to explore the clinical significances of AAA for CRI patients.Results:TYR, PHE and TRP were separated well (R>1.5).The retention time of TYR was approximately 4.3 min, the linear equation was Y=12443.8+58345X, R=0.998, and the limit of detection was 0.014μmol/L for TYR, and the recoveries of TYR were from 98.0%-103.0%, the intra-day and inter-day variations were 3.92%and 4.20%for TYR. The retention time of PHE was approximately 5.8 min, the linear equation was Y=6578.32X+202946.74, R=0.997, and the limit of detection was 0.5μmol/L, and the recoveries of PHE were from 97.8%-104.2%. The intra-day and inter-day variations were 3.14%and 3.37% for PHE.The retention time of TRP was approximately 9.5min, the linear equation was Y=260440.86X-11828.17, R=0.999, and the limit of detection was 0.0049μmol/L, and the recoveries of TRP were from 97.3%-103.5%. The intra-day and inter-day variations were 2.32%and 3.18%for TRP.5-hydroxytryptamine, kynurenine and kynurenic acid have no interferences in this assay, there were good linear relationship betweenthe peaks and concentrations of TYR, PHE and TRP.2. The serum TYR, PHE and TRP concentrations in 100 healthy persons were (63.53±4.68)μmol/L,(66.72±11.57)μmol/Land(44.22±3.67)μmol/L, respectively. The total AAA concentrations were(174.47±11.57)μmol/L and the ratio of TYR to PHE was(0.95±0.40).3. The serum TYR, PHE and TRP concentrations in CRI patients were(52.27±8.25)μmol/L,(61.98±12.38)μmol/L and(21.49±4.25)μmol/L, respectively.The total AAA concentrations were(135.74±12.23)μmol/L. The ratio of TYR to PHE were (0.84±0.67). Compared with healthy groups, there were significantly decreased concentrations of serum AAA,TYR and TRP(t values were 5.095,3.423 and-15.774, P<0.05, respectively. U value was 267.000 for Tyr/Phe).while phe is slightly decrease (t=0.17, p>0.05), there was no statistically significant differences between CRI and healthy group.And there were no statistically significant difference between CRI stages. Chronic nephritis type, DM type and hypertension type patients of CRI, the serum concentrations of TYR, PHE and TRP were significant differences (P <0.05). Serum PHE levels were correlated with the serum UREA and UA, serum TRP levels were correlated with the serum UA and TP. Besides, serum AAA levels were correlated with the serum UA(P<0.05). The total concentrations of serum AAA showed AUC of 0.962 for diagnosis of CRI, when cut off value of the serum AAA was 157.43μmol/L, the diagnostic sensitivity and specificity were 88.9% and 100.0%, respectively.Conclusions:1.A new method was established for simultaneous determination the concentrations of serum PHE, TYR and TRP in healthy persons by high performance liquid chromatography with fluorescence detection.The method is simple, rapid, sensitive and suitable for clinical and scientific study.2.There were clinical significances of AAA for chronic renal insufficiency (CRI) patients.Compared with healthy groups, there were significantly decreased concentrations of serum AAA, TYR and TRP, while PHE is slightly decrease,there was no statistically significant difference between CRI and healthy group.And there were no statistically significant difference between CRI stages.Chronic nephritis type, DM type and hypertension type patients of CRI, the serum concentrations of TYR, PHE and TRP were significant differences.Serum PHE levels were correlated with the serum UREA and UA, serum TRP levels were correlated with the serum UA and TP. Besides, serum AAA levels were correlated with serum UA.And the diagnostic sensitivity and specificity of AAA for CRI were satisfactory. |
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