ABSTRACT
BACKGROUND: 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). METHODS: 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. RESULTS: 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. CONCLUSIONS: The method is simple, fast, accurate, and meets the requirements for simultaneous determination of TRP, KYN and KYNA in serum.
Subject(s)
Chromatography, High Pressure Liquid , Lupus Erythematosus, Systemic/metabolism , Tryptophan/metabolism , Adult , Female , Fluorescence , Humans , Kynurenic Acid/blood , Kynurenine/blood , Lupus Erythematosus, Systemic/diagnosis , Male , Middle Aged , Tryptophan/bloodABSTRACT
OBJECTIVE: To describe a simple, rapid, and sensitive HPLC method for simultaneous determination of TRP and KYNA in human serum. DESIGN AND METHOD: Samples were deproteinized by perchloric acid. KYNA was detected at 344 nm of excitation wavelength and 404 nm of emission wavelength, TRP was detected at 254 nm and 404 nm, with a total run time of 13 min per sample. RESULTS: Standard curves of 0.49 micromol/L to 196 micromol/L of TRP were linear. Inter-day and intra-day coefficient of variations were 3.31% and 4.14%, respectively. Average recovery was 104.43%. Detection limit was 0.001 micromol/L. The linearity of the assay was maintained from 1.5 nmol/L to 2093 nmol/L of KYNA. Inter-day and intra-day CVS were 3.20% and 4.27%, respectively. Average recovery was 101.19%. Detection limit was 0.05 nmol/L. CONCLUSION: The developed HPLC method is simple, convenient and can be applied in the diagnosis of related diseases.
