Determination Of Kynurenic Acid And Tryptophan In Serum By High Performance Liquid Chromatography With Fluorescence Detection And Its Clinical Application

Objective:1. To establish a simple and sensitive method for the simultaneous determination of kynurenic acid (KYNA) and trytophan (TRP) in serum by high performance liquid chromatography with fluorescence detection (HPLC-FLD).2. To detect the KYNA concentration and KA/T ratio in serum of healthy persons and establish the reference values.3. To explore the clinical significance of serum kynurenic acid and tryptophan determined in patients with schizophrenia ( Sch) .Methods:1. Serum samples were deproteinized with 5%(v/v) perchloric acid at equal volum.It employed a Hypersil C8 column and a mobile phase consisted of 0.5 mol/L zinc acetate, 50 mM sodium acetate with 6% (v/v) acetonitrile at a flow rate of 1.5 ml/min. The fluorescence excitation and emission wavelengths were operated at 344 nm and 404 nm respectively at the beginning of the run, and 9.5 minutes later, the excitation wavelength changed to 254 nm.The total assay time of serum was within 14 minutes. 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 KYNA and TRP in healthy persons (n=108) were detected by HPLC-FLD, and the content ratios of KYNA to TRP (KA/T) were calculated. The reference values of KYNA, TRP and KA/T ratio in healthy persons were established.3.The schizophrenia patients were divided into three subgroups (drug-naive, first episode patients, drug-free patients, patients undergoing antipsychotic treatment) and the concentrations of serum KYNA, TRP in the schizophrenia were measured, to explored the clinical significance of serum KYNA and TRP detected in different subgroups of schizophrenia.Results:1. Good linearity was observed in the range of 1.05-2093 nmol/L for KYNA, the lowest detectable limit was 0.05 nmol/L, the average recovery was 101.19%, and the intra-day and inter-day coefficients of variation (CV) were 3.20% and 4.,27% respectively. There also was a good linearity in the range of 0.49-196μmol/L for TRP. The average recovery was 104.43%, the intra-day and inter-day coefficients of variation (CV) were 3.31% and 4.14% respectively. The interferences of PHE, TYR, KYN, 5-HT and Cr were never found with the chromatographic conditions used in this trial.2. The serum KYNA and TRP reference values in 108 healthy persons were 11.85-45.32 nmol/L and 27.15-68.03μmol/L respectively, and the value of KA/T ratio was 0.22-1.03×10^-3 .In 108 healthy persons, TRP level correlated positively with the level of KYNA and age ,with r_s = 0.192, P=0.046 and r_s = 0.247, P = 0.010; meanwhile,KA/T ratio correlated positively also with age(r_s=0.333, P=0.000). And it showed no difference in concentrations of KYNA and KA/T ratio in gender (P>0.05).3. Compared with healthy subjects, there were significantly increased concentrations of serum KYNA and TRP in the drug-naive, first episode patients,and the KA/T ratio were significantly decreased .Conclusions:1. A new method was first established in china for simultaneous determination of KYNA and TRP in serum by HPLC-FLD. The method is simple and rapid, and its precision, sensitivity and accuracy are satisfactory for clinical and scientific study.2.The Chinese adult serum concentration of KYNA has been determined for the first time.3. The concentrations of KYNA and TRP in patients with Sch were different in different subgroups. It showed that antipsychotic treatment may decrease the concentration of KYNA and TRP.