| Objectives:1. To develop a method for simultaneous determination of plasma tryptophan (Trp), kynurenine (Kyn) and 5-hydroxytryptamine (5-HT) by HPLC with programmed wavelength ultraviolet (UV) detection.2. To evaluate the effects of coagulant, anticoagulants and separation gel on determination of Trp, Kyn and 5-HT, and choose the suitable anticoagulant.3. The developed method was applied to determine the concentrations of plasma Trp, Kyn and 5-HT in chronic hepatitis B(CHB)patients, depression patients, uremic patients undergoing hemodialysi(sHD)and their respective controls. Study the metabolism of Trp in patients, and to evaluate the effect of HD in uremic patients.Methods:1. A BDS-Hypersil- C8 column (150 mm×4.6 mm, 5μm) was used for the analysis using theophylline as internal standard (IS) at 25°C. Separation was carried out using the mobile phase consisting of 10 mmol/L acetate buffer (pH 4.5) and acetonitrile (94:6, v/v) at a flow rate of 0.6 mL/min. The eluates were monitored by the programmed wavelength detection setting at 360 nm from 0 to 5.5 min for Kyn, at 220 nm from 5.6 min to 6.0 min for 5-HT, at 302 nm form 6.1 min to 7.0 min for Trp and at 360 nm from 7.1 to 8.0 min for IS.2. Blood was collected into Li-heparin, K2EDTA, K2EDTA with separation gel and coagulants tubes, respectively, and the samples were centrifuged and separated. Then concentrations of Trp, Kyn and 5-HT were determined.3. K2EDTA tube was used to collect blood of fifteen depression patients, twenty uremic patients undergoing HD, One hundred and ten CHB patients and their respective controls, and the samples were centrifuged and separated within 1h of collection. Then concentrations of Trp, Kyn and 5-HT were determined with the present method.Results:1. The retention time of Kyn, Trp, 5-HT and IS were 5.12, 5.81, 6.63 and 7.52 min, respectively. The linearity was from 3.97μmol/L to 400.0μmol/L for Trp, from 0.421μmol/L to 20.2μmol/L for Kyn and from 4.36 nmol/L to 980.5 nmol/L for 5-HT, respectively. The limit of detection were 0.134μmol/L for Trp, 0.016μmol/L for Kyn and 2.03 nmol/L for 5-HT, respectively. The within-day and between-day CVs were less than 4% and 5%, respectively. The mean recovery was in the range of 87 % to 113%.2. Neither K2EDTA with separation gel nor Li-heparin-plasma samples were significantly different from K2EDTA plasma samples. For Trp and Kyn, no significant deviation was observed between the plasma and serum. However, the level of 5-HT was remarkably higher in serum than plasma (p<0.01).3. (1) Levels of Kyn, 5-HT and Trp in plasma of depression patients were significant lower than the controls (p<0.05).(2) Levels of 5-HT, Kyn and the Kyn/Trp ratio were significantly elevated (p<0.01), and Trp levels were reduced in uremic group (p<0.01) compared with the control group. 5-HT, Kyn and the Kyn/Trp ratio have decreased after HD, however, 5-HT levels and the Kyn/Trp ratios remained remarkably higher, and Trp was still lower than in controls.(3) Levels of 5-HT and Trp in CHB patients were significantly lower than in controls (p<0.01), while the Kyn/Trp ratios were remarkably higher than in controls (p<0.01).(4) The logarithm of HBV DNA copies (LogDNA) were significantly positive correlated with Kyn (r=0.547; p=0.000) and Kyn/Trp (r=0.804; p=0.000), and were well negative correlated with 5-HT (r=-0.485; p=0.000) and Trp (r=-0.644; p=0.000). There were also positive associations between the activity of ALT and Kyn (r=0.265; p=0.000), and Kyn/Trp (r=0.224; p=0.020).(5) HBV copies were used to classify the CHB patients to three groups of high (LogDNA>6.0), middle (LogDNA 6.0～4.0) and low (LogDNA<4.0) virus. There were significant deviations between each two groups for Trp and Kyn/Trp (p<0.01). For 5-HT and Kyn, low virus group was significantly different from high and middle virus groups (p<0.01), and there was also significant deviation between high and low virus group (p<0.05).(6) The enzyme activity of ALT (80IU/L) was used to classify the CHB patients to two groups of high and low enzyme activity group. The results showed that there was significant deviation between two groups for Kyn and Kyn/Trp.Conclusions:1. We have developed a method to simultaneously determine Trp, Kyn and 5-HT in plasma by HPLC with programmed wavelength UV detection. The present method is sensitive, rapid, simple and is well suitable for research and clinical routine measurement.2. In this paper, we have studied the metabolism of Trp in depression patients, CHB patients and uremic patients undergoing HD. Result showed that the metabolism of Trp was disturbed in the patients we studied, and it was greatly improved after HD in uremic patients. |
PDF Full Text Request