高效液相色谱-荧光法测定SLE患者血清色氨酸及其代谢产物

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

目的建立一种同时测定血清色氨酸（tryptopban，Trp）、犬尿氨酸（kynurenine，Kyn）和犬尿喹啉酸（kynurenic acid，Kyna）的高效液相色谱-荧光检测法（HPLC—FD），并用此法检测SLE患者血清中Trp及其代谢产物Kyn、Kyna的含量。方法血清标本经0．624mol／L的高氯酸溶液去除蛋白质后取上清液20μl直接进样分析测定。色谱柱为Hypersil C18柱，流动相为0．20mol／L醋酸锌、8．3mmoL／L醋酸和2．5％的乙腈；流速为1．5ml／min；荧光检测激发波长和发射波长在0～11min分别为365nm和480nm，11～15．5min变换为344nm和404nm，15．5～20min为254nm和404nm。采用建立的方法测定体检健康者和SLE患者血清Trp、Kyn和Kyna的含量。结果Trp的线性范围为0．610～196μmol／L，最低检出浓度为0．005μmol／L，平均回收率为103．71％；Kyn线性范围为0．049～98μmol／L，最低检出浓度为0．025μmol/L，平均回收率为97．45％：Kyna线性范围为1．050-1047nmol/L，最低检出浓度为0．050nmol/L，平均回收率为100．60％。日内、日间测定值的变异系数均小于5％。苯丙氨酸、酪氨酸、5-羟色胺对测定均无干扰。与正常对照组比较，SLE患者的Trp含量显著减少，Kyn和Kyna的含量及Kvn／Trp的比值显著增高。结论建立的HPLC—FD法能同时测定血清中Trp、Kyn和Kyna的含量，简便、快速、灵敏、特异。

Objective A method was developed for the simultaneous determination of tryptophan （Trp） and its metabolites kynurenine （Kyn） and kynurenic acid （Kyna） by high performance liquid chromatography with fluorescence detection （HPLC-FD）, and testing serum levels of Trp metabolites in systemic lupus erythematosus （SLE） patients. Methods Serum samples were deproteinized by equal volume of 0.624 mol/L perchloric acid. The analytical column was Hypersil C18 column, and the mobile phase was 0.20 mol/L zinc acetate, 8.3 mmol/L acetic acid, and 2.5% acetonitrile; flow rate was 1.5 ml/min. The excitation and emission wave length of fluorescence detector were 365 nm and 480 nm in 0 - 11 min, 344 nm and 404 nm in 11 - 15.5 min, 254 nm and 404 nm in 15.5 - 20 min, respectively. Results The linear range of Trp was 0. 610 - 196 μmol/L, the detection limit was 0. 005 μmol/L, and the average recovery was 103.71%. The linear range of Kyn was 0.049 -98 μmol/L, the detection limit O. 025μmol/L, and the average recovery was 97.45%. The linear range of Kyna was 1. 050 - 1047 μmol/L, the detection limit was 0.050 nmol/L, and the average recovery was 100.60%. Inter- and intra-day precisions were both less than 5%. Phenylalanine, tyrosine, and 5-hydroxytryptamine had no interference. The assay was employed to analyze serum samples of SLE patients. The result showed significant difference in Trp, Kyn, and Kyna content, Kyn/Trp ratio between SLE patients and control group. Conclusions A new method was established for simultaneous determination of Trp, Kyn, and Kyna in serum. The method is simple, fast, sensitive, specific, and suitable for applicability to clinical measurement.