Simultaneous Determination Of Tryptophan,Kynurenine And Kynurenic Acid Concentrations In Human Serum By HPLC With Fluorescence Detection And Its Clinical Application In SLE

Objective:1. To establish a simple and rapid method for the simultaneous determination of tryptophan(TRP), kynurenine(KYN), kynurenic acid (KYNA) by high performance liquid chromatography with fluorescence detection (HPLC-FLD).3. To explore the clinical significance of serum tryptophan,kynurenine and kynurenic acid in patients with systemic lupus erythematosus (SLE).Methods:1.Serum samples were deproteinized by equal volume of 0.624mol/L perchloric acid.lt employed a Hypersil C18 column and a mobile phase consisted of 0.2 mol/L zinc acetate,8.3 mmol/L acetic acid with 2.5% (v/v) acetonitrile at a flow rate of 1.5 ml/min. The fluorescence excitation and emission wavelengths, respectively, were 365nm and 480nm in 0-11min,344nm and 404nm in 11-15.5 min,254nm and 404nm in15.5-22min. The effects of various aspects on operation and determination were examined to establish optimal assay conditions, such as detection wavelength, the content of organic solvent in the mobile phase, precipitants and flow rate.2. The concentrations of serum TRP,KYN and KYNA in patients with systemic lupus erythematosus (SLE) (n=50) were determined by HPLC-FLD and the content ratios of KYN to TRP (KYN/TRP) and KYNA to KYN (KYNA/KYN)were calculated. We initially explore the clinical significance of changes in their content in differential diagnosis, treatment monitoring, mechanism and so on. Discuss the relation between the serum TRP, KYN, and KYNA and KYN/TRP, KYNA/KYN ratio and disease occurrence and development.Results:1. The retention time of KYN is about 8.5 min, the linear range of KYN was 0.049-98μmol/L, the detection limit 0.025μmol/L, average recovery was 97.45%; The retention time of KYNA is about 13.7 min, the linear range of KYNA was 1.050-1047μmol/L, the detection limit was 0.050nmol/L, average recovery was 100.60%. The retention time of TRP is about 17.6 min, the linear range of TRP was 0.610-196μmol/L, and the detection limit was 0.005μmol/L, average recovery was 103.71%;Inter-day and intra-day precisions were less than 5%. The interferences of PHE, TYR,5-HT and Cr were never found with the chromatographic conditions used in this method.2. SLE patients have lower serum TRP level than healthy control subjects, and higher serum KYN and KYNA levels and higher KYN/TRP. However, there was no difference in KYNA/KYN between SLE and healthy control subjects.Conclusions:1. A new method was established for simultaneous determination of TRP,KYN and KYNA in serum by high performance liquid chromatography with fluorescence detection (HPLC-FLD). The method is simple and rapid, and its precision, sensitivity and accuracy are satisfactory for clinical and scientific study.2. The concentrations of TRP,KYN and KYNA in patients with SLE altered significantly. There was severe metabolite disorder of serum tryptophan in SLE patients. It suggested that Simultaneous determination of serum TRP, KYN and KYNA contents in SLE patients not only provides a measure of TRP metabolism for potential clinical diagnosis but can also monitor immune response in vivo.