Simultaneous determination of serum tryptophan metabolites in patients with systemic lupus erythematosus by high performance liquid chromatography with fluorescence detection

Abstract

To provide a more comprehensive clinic marker of tryptophan (TRP) catabolism in patients with systemic lupus erythematosus (SLE), we developed a simple and efficient method that simultaneously measured serum TRP, kynurenine (KYN), and kynurenic acid (KYNA) using high performance liquid chromatography with fluorescence detection (HPLC-FD). A simple and specific high performance liquid chromatography (HPLC) method was developed for simultaneously quantitative determination of TRP, KYN and KYNA with fluorescence detection (FD) using programmed wavelength and on-column fluorescence derivatization. Thirty patients with SLE and 80 healthy control subjects were analyzed for serum TRP metabolites using the assay we developed. The tryptophan breakdown index (TBI) and neuroprotective ratio (NPR) were calculated. The retention time of KYN, KYNA and TRP were 8.5 min, 13.7 min and 17.6 min, respectively. The linear range for TRP was 0.245-196 micromol/L, the limit of detection (LOD) was 0.001 micromol/L and average recovery was 103.71%. The linear range for KYN was 0.049-98 v/L, the LOD was 0.0245 micromol/L, and average recovery was 97.45%. The linear range for KYNA was 1.05-2093 nmol/L, the LOD was 0.05 nmol/L, and average recovery was 100.60%. Inter-day and intra-day relative standard deviations (SDs) were <5%. Phenylalanine, tyrosine, 5-hydroxytryptamine and creatinine did not interfere with the method. The results showed great differences in TRP, KYN and KYNA contents and TBI between patients with SLE and healthy controls, but little difference in NPR. The method is simple, fast, accurate, and meets the requirements for simultaneous determination of TRP, KYN and KYNA in serum.