【作者】 穆萨；

【导师】 唐爱国；

【作者基本信息】 中南大学 ， 临床检验诊断学， 2011， 硕士

【摘要】 目的：1.建立一种简单、快速并能同时测定血清中酪氨酸(Tyr)、5-羟色胺(5-HT)和色氨酸(Trp)的高效液相色谱—荧光检测法(HPLC-FLD)。2.初步探讨血清酪氨酸(Tyr)、5-羟色胺(5-HT)和色氨酸(Trp)含量测定在重性抑郁障碍(MDD)患者中的临床价值。方法：1.血清标本与等量5%(V/V)高氯酸溶液混合处理去蛋白后离心取上清液20μL直接进样分析。用Atlantis C18 column (4.6×150mm,i.d.5μm)分离；流动相为0.1 mol/L KH2P04:甲醇(85：15,V/V)；流速1.0mL/min;荧光检测波长：0-4min激发光波长228nm,发射光波长为306mn;4-7.5min激发光波长278nm,发射光波长为338nm;7.5min后激发光波长285nm,发射光波长为353nm。室温下12min内完成分析测定。Tyr.5-HT和Trp均采用峰保留值比较法和叠加法进行定性分析,用外标法测定峰面积进行定量分析。通过对线性范围和检测限、精密度、回收率和干扰试验的考察,对该方法进行方法学评价。2.测定健康成人(n=110例)和MDD患者(n=60例)血清Tyr、5-HT和Trp含量。探讨MDD患者血清Tyr、5-HT和Trp含量含量变化临床意义。结果：1.血清和标准品样本的Tyr、5-HT和Trp峰均分离良好,保留时间分别约为3min、5min和9min。Tyr的线性范围为0.55-275μmol/L,最低检出浓度为0.014μmol/L,回收率为98.0%-103.3%；5-HT的线性范围为0.094-47.03μmol/L,最低检出浓度为0.024μmol/L,回收率为96.3%-103.1%；Trp的线性范围为0.049-49μmol/L,最低检测浓度为0.0049μmol/L,回收率为93.3%-103.5%。Tyr、5-HT和Trp的日内、日间测定的相对标准偏差均小于5%。苯丙氨酸(Phe)、犬尿喹啉酸(Kyna)、犬尿氨酸(Kyn)和肌酐(Cre)等物质对该法均无干扰。2.110例健康成人血清Tyr含量为(63.68±2.23)μmol/L,血清5-HT含量为(1.15±0.04)gmol/L,血清Trp含量为(42.46±1.17)μmol/L, 5-HT/Trp(×10-3)比值为(26.20±7.21)。与正常对照组比较,MDD患者组血清Tyr、5-HT和Trp含量显著降低(P<0.01)。Tyr诊断MDD的敏感性和特异性分别为：85.0%和100.0%；5-HT/Trp比值诊断MDD的敏感性和特异性分均为100%。结论：1.本文在国内外首次建立HPLC-FLD法同时测定血清Tyr、5-HT和Trp,方法简单、快速、灵敏、特异、准确,适宜于临床和科研工作。2.血清Tyr、5-HT和Trp对MDD的临床辅助诊断有一定的临床价值。更多还原

【Abstract】 Objective:1. To establish a simple and rapid method for simultaneous determination of tyrosine (Tyr),5-hydroxytryptamine (5-HT), and tryptophan (Trp) in serum by high performance liquid chromatography-fluorescence detection (HPLC-FD).2. To explore the clinical significance of serum Tyr,5-HT, and Trp in patients with major depressive disorder (MDD).Methods:1. A 20μL supernatant of a serum sample deproteinized by 5% perchloric acid solution was assayed and separated on an Atlantis C18 column (4.6x150mm, i.d.5μm). Best results were achieved with an elution of 0.lmol/L KH2PO4 and methanol (85:15, V/V), a flow rate of 1.0mL/min. The fluorescence was recorded at the optimal wavelength for Tyr (λex=228nm,λem=306nm) from 0 to 4 min,5-HT(λex=278nm,λem=338nm) from 4 to 7.5 min and the optimal wavelength for Trp (λex =285nm,λem=353nm) after 7.5 min. The total assay time of serum was within 12 minutes. The presence of Tyr,5-HT, and Trp in serum was determined by comparing their peak retention times with those of external standards.2. The concentrations of Tyr,5-HT, and Trp in healthy controls (n=110) were determinate by HPLC-FD. The concentrations of Tyr and Trp in MDD patients (n=60) were determinate, to explore the clinical significance of serum Tyr,5-HT, and Trp in patients with MDD.Results:1. Under optimal conditions excellent linearity was obtained in the range of 0.55-275μmol/L,0.094-47.03μmol/L, and 0.049-49μmol/L for Tyr,5-HT, and Trp, and the detection limits were 0.014μmol/L,0.024μmol/L, and 0.0049μmol/L for Tyr,5-HT and Trp, and the recovery was 98.0%-103.3%,96.3%-103.1%, and 93.3%-103.5% for Tyr,5-HT and Trp, respectively. The precision results showed that the relative standard deviation of intra-day and inter-day were<5%, respectively. The results indicated Phe, Kyna, Kyn, and Cre had no interfering effect to the determination of Tyr,5-HT, and Trp.2. The concentrations of Tyr,5-HT, and Trp in healthy controls were (mean±SD,63.68±2.23)μmol/L, (mean±SD,1.15±0.04)μmol/L, and (mean±SD,42.46±1.17)μmol/L, respectively. The ratio of 5-HT/Trp(x10-3)was(mean±SD,26.20±7.21). The concentrations of Tyr, 5-HT, and Trp were significantly lower in patients with MDD than in healthy controls (54.02±9.43μmol/L vs.63.68±2.23μmol/L, 0.28±0.19μmol/L vs.1.15±0.04μmol/L,and 39.00±7.23μmol/L vs. 42.46±1.17μmol/L for Tyr,5-HT, and Trp, respectively; p<0.01). In addition, Tyr as an index on the MDD diagnostic sensitivity and specificity were 85.0% and 100.0%, and the ratio of 5-HT/Trp on the MDD diagnostic both sensitivity and specificity were 100.0%.Conclusions:1. The present method by HPLC with programmed fluorescence wavelength detection is simple, rapid and sensitive as compared to other conventional methods of separate or simultaneous determination of Tyr,5-HT, and Trp in serum and will be suited for high throughput screening. The described method is simple, fast, accurate, and suitable for routine analysis of serum Tyr,5-HT and Trp in serum.2. It is of great value to determination of Tyr,5-HT, and Trp for defining clinical staging and identifying pathologic category of MDD.更多还原