| Objective:1.To establish a simple and rapid method for the determination of Kynurenine(KYN)in serum by high performance liquid chromatography fluorescence detection(HPLC-FLD)with on-column derivatization.2.To establish a method for the determination of Kynurenine(KYN) and trytophan(TRP)in serum by HPLC-FLD with on-column derivatization.3.To explore the clinical significance of serum Kynurenine and tryptophan in patients with Rheumatoid Arthritis.Methods:1.Serum samples were deproteinized by equal volume of 5%(v/v) perchloric acid.It employed a Hypersil C8 column and a mobile phase consisted of 0.25 mol/L zinc acetate,50 mM acetic acid with 3%(v/v) acetonitrile at a flow rate of 1.5 ml/min.The fluorescence excitation and emission wavelengths were operated at 365 nm and 480 nm respectively. 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 method established was improved by adjusting fluorescence excitation and emission wavelengths.At the beginning of the run, fluorescence excitation and emission wavelengths were operated at 365 nm and 480 nm respectively and the excitation and emission wavelength changed to 254 nm and 404nm after 10 minutes.3.The concentrations of serum KYN and TRP in patients with rheumatoid arthritis(n=110)were determined by HPLC-FLD and the content ratios of KYN to TRP(KYN/TRP)were calculated.The RA patients were divided into four groups according modified disease activity score to explore the relation between the serum KYN,TRP and disease activity.Results:1.Good linearity was observed in the range of 0.098～19.6μmol/L for KYN,the lower limit of detection was 0.04μmol/L,the recovery was 90.8-96.2%,and the intra-day and inter-day coefficients of variation(CV) were 3.82%and 4.63%respectively.The interferences of PHE,TYR, KYNA,5-HT and Cr were never found with the chromatographic conditions used in this method.2.There also was a good linearity in the range of 4.9～196μmol/L for tryptophan.The recovery was 92.6-106.9%,the intra-day and inter-day coefficients of variation(CV)were 3.63%and 4.44% respectively.The interferences of PHE,TYR,KYNA,5-HT and Cr were not found with the chromatographic conditions used in this method. 3.Compared with healthy subjects,there were significantly increased concentration of serum KYN and significantly decreased concentrations of TRP in the patients with RA.Positive relation was founded between the serum KYN,KYN/TRP and disease activity.Conclusions:1.A new method was established for simultaneous determination of KYN in serum by high performance liquid chromatography fluorescence detection(HPLC-FLD)with on-column derivatization.The method is simple and rapid,and its precision,sensitivity and accuracy are satisfactory for clinical and scientific study.2.A new method was established for simultaneous determination of KYN and TRP in serum by HPLC-FLD with on-column derivatization.3.The concentrations of KYN and TRP in patients with RA altered significantly.There was severe unbalance of Kynurenine pathway in peripheral tissues in RA patients.It suggested that KYN/TRP Ratio is positive relation to disease activity and was marker of disease activity.
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