Determination Of Kynurenine And Kynurenic Acid In Serum By High-performance Liquid Chromatography With Fluorescence Detection And Its Clinical Application

Objective:1. To establish a simple and rapid method for simultaneous determination of kynurenine (KYN) and kynurenic acid (KYNA) in serum by high performance liquid chromatography with fluorescence detection (HPLC-FLD).2. To investigate the effect of sample collection and storage conditions on KYN and KYNA measurements in blood samples.3. To establish reference values of kynurenine (KYN), kynurenic acid (KYNA) and KYNA/KYN ratio in serum of healthy adult.4. To explore the clinical significance of serum KYN and KYNA in patients with chronic renal failure (CRF).Methods:1. Supernatant fluid of serum precipitated with perchloric acid was isocratically eluted using Hypersil C8 column and a mobile phase consisted of 0.25mol/L zinc acetate, 50 mmol/L acetic acid with3% (v/v) acetonitrile at a flow rate of 1.5ml/min. The excitation and emission wavelengths were operated at 365 nm and 480 nm respectively at the beginning of the run, and 10 minutes later, the excitation and emission wavelength changed to 344 nm and 404nm. The total assay time of serum was within 15 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 and flow rate.2. The concentration of kynurenine and kynurenic acid in plasma and serum samples preserved under different conditions were determind by HPLC-FLD.3. The concentrations of serum KYN and KYNA in healthy controls (n=71) were determined by HPLC-FLD, and the content ratio of KYNA to KYN (KYNA/KYN) were calculated. The reference values of KYN, KYNA and KYNA/KYN ratio in healthy adults were established.4. The concentrations of serum KYN and KYNA in CRF patients (n=76) were determined, to explored the clinical significance of serum KYN and KYNA determined in different progresses of CRF.Results:1. Good linearity was observed in the range of 0.098～19.6μmol/L for KYN, the limit of detection was 0.05μmol/L, the average recovery was 94.88%, and the intra-day and inter-day coefficients of variation (CV) were 3.87% and 3.94% respectively. There also was a good linearity in the range of 2.62～1047nmol/L for kynurenic acid, the limit of detection was 0.11nmol/L; The average recovery was 102.72%, the intraday and interday coefficients of variation (CV) were 3.79% and 4.71% respectively. The interferences of PHE, TYR, TRP, 5-HT and Cr were never found with the chromatographic conditions used in this trial.2. There is no statistical significance between EDTA-K2 anticoagulant plasma and serum ( P>0.05). The change of KYNA and KYN concentration in serum samples kept at -20℃for a week was not significant (P> 0.05). KYN and KYNA decreased when samples were preserved at 4℃for a week,but the change was not significant. KYN was stable whitin 72 hour at room temperature and KYNA was stable whitin 120 hour at room temperature.3. The serum KYN and KYNA levels in 71 healthy persons were 0.95～2.82μmol/L (x|-±s=1.40±0.34μmol/L), 11.79～46.69 nmol/L (x|-±s =24.22±8.67 nmol/L) and the levels of KYNA/KYN ratio were 11.76～28.36 nmol/μmol (x|-±s =29.271±9.315 nmol/μmol). In different age groups, it showed no difference on the concentration of KYN, KYNA and KYNA/KYN ratio in serum.4. Compared with the control group, CRF serum KYN and KYNA was significantly higher (P <0.01); CRF patients at different stages of serum KYN and KYNA content with the condition and the development of higher (P <0.01); serum and CRF KYN and KYNA and renal function in a certain relevance.Conclusions:1. A new method was established for simultaneous determination of KYN and KYNA in serum by HPLC-FLD. The method is simple and rapid, and its precision, sensitivity and accuracy are satisfactory for clinical and scientific study. The reference values of concentrations of serum KYN,KYNA and KYNA/KYN ratio can provide parameters to study of corresponding diseases.2.There is no statistically significance between EDTA-K2 anticoagulant plasma and serum . Serum samples at room temperature (25℃±1) under stable conditions for three days, if not promptly detected, it is best placed≤-20℃under the conditions of preservation.3. It is the first time in China to simultaneously determine the concentration of KYN and KYNA, KYNA / KYN ratio in serum of health adult using HPLC-FLD.4. The concentrations of KYN and KYNA in patients with CG and CRF altered significantly. It showed there was severe unbalance of kynurenine pathway in CRF patients.