高效液相色谱法在类风湿关节炎患者血清TRP和KYN检测中的应用与临床价值

目的 探讨高效液相色谱荧光检测法(HPLC-FLD)同时测定血清色氨酸(TRP)和犬尿氨酸(KYN)对诊断类风湿关节炎(RA)的临床意义.方法 血清标本加等量5%(V/V)高氯酸溶液去除蛋白,离心取上清液20 μl,直接进样分析.色谱柱为HpemilC_8柱(300 mm×6.0 mm i.d,10 μm);流动相为0.25 mol/L醋酸锌和50 mmol/L醋酸溶液(含3%乙腈),流速为1.5 ml/min;0～10 min荧光检测器的激发波长和发射波长分别为365 nm和480 nm,10 min后激发波长和发射波长分别变换为254 nm和404 nm.同时,用该方法测定120名健康成人和110例RA患者血清TRP、KYN和TRP/KYN比值(K/T),并评价其诊断RA的敏感度、特异度和方法学效能.结果 血清标本的KYN和TRP保留时间分别为8.1和11.5 min,两者分离良好.KYN线性范围为0.098 ～19.600 μmol/L,最低检测浓度为0.04 μmol/L;回收率为90.8%～96.2%,日内变异系数为3.68%,日间变异率为4.97%.TRP的线性范围为4.9～196.0μmol/L.最低检测限为0.005 μmol/L,回收率为92.6～106.9%,13内变异系数为3.63%,日间变异系数为4.44%.在本试验的色谱条件下测定苯丙氨酸(Phe)、酪氨酸(Tyr)、犬尿喹啉酸(KYNA)、5-羟色胺(5-HT)和Cr均无干扰.RA组患者血清KYN含量和K/T比值[(2.06±0.38) μmol/L和(55.46±5.81)×10~(-3)]与健康对照组[(1.51±0.35)μmol/L和(32.54 ±9.00)×~(-3)]比较,均显著升高(U=3 251.0,t=10 741,P均为0.000),而RA组TRP含量[(38.24±5.27)μmol/L]与健康对照组[(47.52±5.79)μmol/L]比较,则显著降低(t=10.399,P=0.000).K/T比值诊断RA的敏感度、特异度分别为83.6%(92/110)、85.8%(103/120).结论 HPLC-FLD同时测定血清TRP和KYN的方法精密度、回收率、抗干扰能力、线性范围等均符合临床检测要求.K/T比值可作为RA的辅助性诊断指标.

Simultaneous determination of kynurenine and tryptophan concentrations in serum of RA patients by HPLC fluorescence detection

Objective To explore the clinical significance of serum kynurenine and tryptophan by HPLC fluorescence detection in patients with rheumatoid arthritis ( RA). Methods 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 mmol/L 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 at the beginning of the run, and 10 minutes later, the excitation and emission wavelength changed to 254 nm and 404 nm. TRP and KYN of health group and RA group were determined by HPLC-FLD. Results The retention time of KYN was 8. 1 min;the linearity was from 0. 098 μmol/L to 19. 600 μmol/L with the detection limit of 0. 04 μmol/L , the recovery were 90. 8%-96. 2% , and the intra-day and inter-day variations were 3. 68% and 4. 97% respectively. The retention time of TRP was 11. 5 min, the linearity of the assay was from 4. 9 μmol/L to 196. 0 junol/L, the detection limits was 0. 005 μmol/L,the recovery of TRP was 92. 6% -106. 9% , the intraday and interday coefficients of variations were 3. 63% and 4.44% .respectively. Phenylalanine, tyrosine, serotonin, kynurenic acid and creatinine didn't interfere in the assay. Compared with healthy subjects, there were significantly increased concentration of serum KYN and K/T Ratio [(1.51 ±0.35) nol/L vs(2. 06 ±0. 38) ujnol/L,tf = 3 251. 0,P =0. 000; (32. 54 ± 9. 00) × 10~(-3) vs(55. 46 ±5. 81) × 10~(-3) ,t = 10 741 ,P =0. 000] and significantly decreased concentrations of TRP [(47.52±5.79) μmol/L vs (38.24±5.27) μmol/L; t = 10. 399,P =0. 000) ] in the patients with RA. The sensitivity, specificity of K/T ratio for diagnsis RA was 83. 6% (92/110) and 85. 8 ( 103/ 120)% respectively. Conclusions A new method is established for simultaneous determination of KYN and TRP in serum by HPLC-FLD with on-column derivatization. The method is simple and rapid, and its precision, sensitivity and specificity are satisfactory for clinical and scientific study. K/T Ratio is a supporting diagnosis biomarker of RA.