【作者】 马莉；

【导师】 丁敏；

【作者基本信息】 重庆医科大学 ， 临床检验诊断学， 2010， 硕士

【摘要】 目的:1.建立一种同时测定血浆中色氨酸(tryptophan,Trp)与犬尿氨酸(kynurenine,Kyn)的高效液相色谱程序波长设计紫外检测方法(HPLC-VWD))。2.用HPLC-VWD内标法测定正常人和慢性乙肝患者血浆中Trp与Kyn浓度,探讨乙肝患者Trp代谢的变化。3.建立一种高效液相色谱-荧光检测法(HPLC-FD)同时测定血浆中谷胱甘肽(glutathione,GSH)、半胱氨酸(cysteine,Cys)、同型半胱氨酸(homocysteine, Hcy)、半胱氨酰甘氨酸(cysteinylglycine,CysGly)四种硫醇物浓度并对它们各自的总量、还原型和游离型分别定量。4.利用主成份分析(principal components analysis,PCA)研究尿毒症患者透析前后血浆硫醇物的代谢。5.利用偏最小二乘法判别分析(partial least-squares discriminant analysis, PLS-DA)及其载荷图研究血浆硫醇物对尿毒症的诊断能力。方法:1.以3-硝基酪氨酸(3-nitro-tyrosine,3-N-Tyr)为内标,采用Agilent Hypersil ODS (125.0 mm×4.0 mm,5μm)分离。流动相为醋酸缓冲液(15 mmol/L, pH 5.5):乙腈为94:6(v/v);流速为0.8 mL/ min;柱温:25℃;紫外程序化检测波长:0～4.0 min, 360 nm; 4.1～5.0 min, 302 nm。用此方法测定15例正常人和15例慢性乙肝患者血浆中Trp与Kyn。2.以tris-(2-carboxylethyl)-phosphine (TCEP)为还原剂,N-(1-pyrenyl) maleimide (NPM)为衍生剂,采用Agilent Hypersil ODS (250.0 mm×4.0 mm,5μm)分离。流动相A:15 mmol/L醋酸盐溶液,流动相B为乙腈,流动相C为水溶液(300 ml水中加入1 ml醋酸和1 ml磷酸),梯度洗脱。柱温:25℃,流速:0.5 ml/min,检测波长:激发波长330 nm,荧光波长380 nm,进样量:20μl。用所建立的方法检测28例健康人和29例尿毒症患者血液透析前后血浆中总量、游离型和还原型GSH、Cys、Hcy、CysGly浓度。结果:1. Kyn与Trp的保留时间分别为2.9 min和4.4 min,线性范围分别为0.442μmol/L ~18.3μmol/ L和3.67μmol/L ~470μmol/L,检测限分别为0.014μmol/L和0.122μmol/L。Kyn与Trp的日内变异均小于3%,日间变异均小于4%。平均回收率均在92.29%与104.4%之间。慢性乙肝组Kyn/Trp明显高于对照组。2. GSH、Cys、Hcy和CysGly总量的线性范围分别为2.00~80.0μmol/L,10.0~1500μmol/L,1.00~120μmol/L,3.00~240μmol/L,r为0.9990～0.9999,具有良好的相关性。GSH、Cys、Hcy和CysGly还原型的线性范围分别0.10~8.00μmol/L,1.25~50.0μmol/L,0.01~4.00μmol/L,0.05~6.00μmol/L,r为0.9992～0.9996,具有良好的相关性。最低检测限为0.005～3.0μmol/L。所测硫醇物浓度,日内精密度不超过5.0%,日间精密度均小于9.0%。平均回收率为80%～115%。3.①尿毒症患者透析前血浆中Hcy、Cys和、CysGly总量、游离和还原型浓度高于正常组,GSH总量、游离和还原型浓度低于正常组。②尿毒症患者透析后血浆中各种形式的GSH均较正常组低。总量与游离型Cys透析后降至正常,但还原型Cys仍高于正常。Hcy总量和还原型透析后降至正常但游离型高于正常。透析后各种形式的CysGly浓度均高于正常组。③尿毒症患者血浆总量、游离和还原型Cys和Hcy浓度透析后比透析前分别降低。总量Cys和Hcy浓度分别降低了56.5%、53.6%;游离型Cys和Hcy浓度分别降低了81.5%、48.1%;还原型Cys和Hcy浓度分别降低了23.8%、51.9%。血浆总量、游离和还原型GSH和CysGly浓度透析前后无明显变化。④主成分得分图显示透析前与透析后和正常组基本区分,透析前相对分散,透析后相对集中且更接近于正常组。⑤运用PLS-DA模型诊断尿毒症透析前患者,正确率为93.1%;诊断透析后患者,正确率为82.8%;其总的判对率为89.3%。结论:1.本研究建立了一种HPLC-VWD检测法同时测定血浆中Kyn和Trp,采用内标法定量。本方法操作简便,分析时间短,内源性物质干扰小,特异性好,准确度高,适合于临床检验。2.本文建立了以NPM为衍生剂,TCEP为还原剂的硫醇物柱前衍生荧光检测法。该方法不仅可同时测定tGSH、tCys、tHcy和tCysGly,还可测定他们各自的游离型和还原型。该方法简单、快速、灵敏。3.本研究发现尿毒症患者硫醇物的代谢发生了紊乱。透析对尿毒症患者硫醇物的代谢紊乱的纠正有明显效果。建立PLS-DA模型能很好的判别透析前患者、透析后患者和正常对照组。载荷图表明rHcy、fHcy和tHcy与透析前的尿毒症患者密切相关且rHcy对尿毒症的诊断更有价值。更多还原

【Abstract】 Objective:1. To established a method for simultaneous determination plasma kynurenine (Kyn) and tryptophan (Trp) by HPLC with variable wavelength detection (HPLC-VWD).2. The developed method was applied to determination of plasma Trp and Kyn concentration in hepatitis B virus (HBV) subjects and investigated the change of Trp catabolism while HBV infection.3. To establish a method simultaneously determination total concentrations of homocysteine (Hcy), cysteine (Cys), Cysteinylglycine (CysGly) and glutathione (GSH) with fluorescence detection (FD) using N-(1-pyrenyl) maleimide (NPM) as derivatization and their total, reduced, free fractions were also measured.4. The developed method was applied to determination of plasma thiols concentration in uremia patients before hemodialysis (B HD) and after hemodialysis (A HD) .The metabolism of plasma thiols in uremia patients was ananlyzed by principal components analysis (PCA).5. The partial least-squares discriminant analysis (PLS-DA) and loading plots were analyzed for the ability of plasma thiols fraction with high discriminating power, that is, potential biomarker.Methods:1. Kyn and Trp were separated on Agilent Hypersil ODS column (125.0 mm×4.0 mm,5μm) using 3-nitrotyrosine as internal standard. The mobile phase consisted of 15 mmol/L sodium acetate-acetic acid(pH 5.5)containing 6 %( v/v) acetonitrile at a rate of 0.8 ml/min. The chromatographic separation was performed at 25℃.The eluate was monitored by the programmed wavelength detection setting at 360 nm from 0 to 4.0 min for Kyn and at 302 nm from 4.1 min to 5.0 min for Trp. The developed method was applied to determination of plasma Trp and Kyn in hepatitis B virus (HBV) subjects.2. Plasma thiols were separated on Agilent Hypersil ODS column (250.0 mm×4.0 mm,5μm) using TCEP as reduce regent and NPM as derivatization. The mobile phase consisted of A (15 mmol/L sodium acetate), B(acetonitrile) and C(300 ml water containing 1 ml acetic acid and 1 ml phosphoric acid) at a rate of 0.8 ml/min. The chromatographic separation was performed at 25℃. The eluate was monitored by fluorescence detection withλex at 330 nm andλem at 380 nm. The developed method was applied to determination of plasma total, free and reduced thiols in B HD and A HD of uremia patients.Results:1. The retention time of Kyn and Trp were 2.9 min and 4.4 min, respectively. For Kyn, the assay was linear from 0.442μmol/L to 18.3μmol/L. For Trp, the linearity was from 3.67μmol/L to 470μmol/L. The detection limits were 0.014μmol/L for Kyn and 0.122μmol/L for Trp, respectively. The within-day and between-day CVs were less than 3% and 4%, respectively. The mean recovery was in the range of 92.29 % to 104.4%.2. The linearities of the assay for total GSH, Cys, Hcy and CysGly were 2.00~80.0μmoL/L, 10.0~1500μmoL/L, 1.00~120μmoL/L and 3.00~240μmoL/L, respectively. For reduced GSH, Cys, Hcy and CysGly , the linearities were 0.10~8.00μmol/L, 1.25~50.0μmol/L, 0.01~4.00μmol/L and 0.05~6.00μmol/L, respectively and the regressions ranged from 0.9992 to 0.9996. The detection limit ranged from 0.005 to 3.0μmol/L. The within-day and between-day CVs were less than 5% and 9%, respectively. The mean recovery was in the range of 80 %to 115%.3.①There were increase in total, free and reduced Hcy, Cys and CysGly in patients before hemodialysis compared to healthy subjects while GSH was lower than healthy subjects.②Plasma most thiols were also abnormal in patients after hemodialysis. But plasma total, free Cys and total, free Hcy were reduced to normal after dialysis.③There were decrease in total, free, reduced Hcy and Cys in uremia patients before dialysis than after dialysis,while there were no significant change in the three fractions of GSH and CysGly concentrations.Conclusion:1. We have developed a new method for simultaneous determination of Kyn and Trp in plasma by HPLC-VWD. The method is simple and rapid, and its precision, sensitivity and accuracy are satisfactory. The presented method is well suitable for clinical routine measurement.2. In this paper, we describe an HPLC method for simultaneously determination total, free and reduced of GSH, Cys, Hcy and CysGly with fluorescence detection using NPM as derivatization. The developed method is fast, simple, sensitive and precise.3. This research found the metabolism of plasma thiols was abnormal. The PCA scatter plots showed the B HD could be apart from A HD and control group. Hemodialysis has effects on the correction of plasma thiols metabolism abnormal in uremia. The loading plots of total, free and reduced Hcy were analyzed. We found the ability of reduced fraction with high discriminating power, that is, potential biomarker.更多还原