高效液相色谱测定血浆中氨基酸的方法学研究及临床应用
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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。
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.
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。
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.
引文
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[41] Wang R, Tang AG. Simultaneous Determination of Kynurenine and Tryptophan in Serum by High Performance Liquid Chromatography [J]. Chin J Chromatogr. 2006, 24: 140-143
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[49] Ueland PM, Refsum H, Stabler SP, et al. Total homocystine in plasma or serum methods and clinical applications [J]. Clin Chem. 1993, 39: 1746-1749
[50] Ramussan K, Moller J, Lyngbak M. Mithin-person variation of plasma homocysteine and effects of posture and tourniquet application [J]. Clinical Chemistry. 1999, 45(10): 1850-1855
[51] Andersson A, Lsaksson A, Hultberg B. Homocysteine export from erythrocytes and its implication for plasma sampling [J]. Clin Chem. 1992, 38: 1311-1315
[52] Ducros V, Candito M, Causse E, et al. Plasma homocysteine measurement: a study of preanalytical variation factors for conditions for total plasma homocysteineconcentrations [J]. Ann Biol Clin. 2001, 59: 33-39
[53] Theoret Y, Rivard G E, Infante Rivard C, et al. Stability of plasma homocysteine in perinatology [J]. Clinica Chimica Acta. 2002, 319: 63-66
[54] Moller J, Rasmussen K. Homocysteine determination [J]. Clin Chem Acta. 2001, 309: 53-56
[55] Miner S E S, Evroski J, Cole D E C. Theclinical chemistry and molecular biology of homocysteine metabolism: an update [J]. Clin Biochem. 1997, 30: 189-201.
[56] Diane M H, Lisa J J. Effect of temperature on stability of blood homocysteine in collection tubes containing 3-deazaadenosine [J]. Clinical Chemistry. 2002, 48(11): 2017-2022.
[57] Brandl R, Probst R, Muller B, et al. Evaluation of the measurement of homocysteine in patients with symptomatic arterial disease and in healthy volunteers[J]. Clin Chem. 1999, 45: 699-702.
[58] Arnadottir M , Berg AL , Hegbrant J , et al. Influence of haemodialysis on plasma total homocysteine concentration [J ] . Nephrol Dial Transplant. 1999, 14(1):142-145
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[16] Pertovaara M, Raitala A, Uusitalo H. Mechanisms dependent on tryptophan catabolism regulate immune responses in primary Sj?gren's syndrome [J]. Clin Exp Immunol. 2005, 142: 155-161
[17] Laich A, Neurater G, Widner B, et al. More rapid method for simultaneous measurement of tryptophan and kynurenine by HPLC [J]. Clin Chem. 2002, 48: 579-581
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[20] Marklova E, Makovickova H, Krakorva I. Screening for defects in tryptophan metabolism [J]. J Chromatogr. 2000, 870: 289-293
[22] Suliman M E, Anderstam B, Lindholm B. Total, free, and protein-bound sulphur amino acids in uraemic patients [J]. Nephrol Dial Transplant. 1997, 12: 2332–2338
[23] Mansoor MA, Ueland PM, Aarsland A, ea tl. Redox status and protein binding of plasma homocysteine and other aminothiols in patients with homocystinuria [J].Metabolism. 1993, 41:1481–1485
[24] Hoffer L J, Robitaille L, Ellan K M, et al. Plasma reduced homocysteine concentrations are increased in end-stage renal disease [J]. Kidney International. 2001, 59: 372–377
[25] Chambers J C, Ueland P M, Wright M, et al. Investigation of relationship between reduced, oxidized, and protein-bound homocysteine and vascular endothelial function in healthy human subjects [J]. American Heart Association. 2001,89:187-192
[26] Yang T H, Chang C Y, Hua M L, Various forms of homocysteine and oxidative status in the plasma of ischemic-stroke patients as compared to healthy controls [J]. Clinical Biochemistry. 2004, 37: 494– 499
[27] Sj?berg B, Bj?rn A, Mohamed S. Plasma reduced homocysteine and other aminothiol concentrations in patients with CKD [J]. American Journal of Kidney Diseases. 2006, 47(1): 60-71
[28] Frantzen F, Faaren A L, Alfheim L, et al. Enzyme conversion immunoassay for determining total homocysteine plsma or serum [J]. Clin Chem. 1998, 44: 211-216.
[29] Yu H H, Joubran R, Asmi M, et al. Agreement among four homocyseine assays and results in patients with coronary atherosclerosis and controls [J]. Clin Chem. 2000, 46: 258-264.
[30] Martin S C, Tsakas-Ampatzis L, Bartlet WA, et al. Measurement of total plasma homocysteine by HPLC with coulometric detection [J]. Clin Chem. 1999, 45: 150-152
[31] Sigit J I, Hages M, Brensing K A, et al. Total plasma homocysteine and related amino acids in end-stage renal disease (ESRD) patients measured by gas chromatography-mass spectrometry comparison with the abbott IMx homocysteine assay and the HPLC method [J]. Clin Chem Lab Med. 2001,39 (8): 681-690
[32] Madamanchi N R, Vendrov A, Runge M S, et al. Oxidative Stress and VascularDisease [J]. Thromb Vasc Biol. 2005, 25: 29-38
[33] Bald E, Chwatko G, G?owacki R, et al. Analysis of plasma thiols by high-performance liquid chromatography with ultraviolet detection [J]. Journal of Chromatography A, 2004, 1032: 109–115
[34] Chassaing C, Gonin J, Wilcox C S, et al. Determination of reduced and oxidized homocysteine and related thiols in plasma by thiol-specific pre-column derivatization and capillary electrophoresis with laser-induced fluorescence detection. Chromatogr B. 1999, 735 (4): 219-227
[35] Nolin T D, McMenamin M E, Himmelfarb J. Simultaneous determination of total homocysteine, cysteine, cysteinylglycine, and glutathione in human plasma by high-performance liquid chromatography: Application to studies of oxidative stress [J]. Journal of Chromatography B. 2007, 852: 554–561
[36] Benkova B, Lozanov V, Ivanov I P, et al. Determination of plasma aminothiols by high performance liquid chromatography after precolumn derivatization with with N-(2-acridonyl)maleimide. Journal of Chromatography B. 2008, 870: 103-108
[37] Wald D S, Law M, Morris J K. Homocysteine and cardiovascular disease: evidence on causality from a meta-analysis [J]. BMJ. 2002, 325: 1202-1208
[38] Boysen G, Brander T, Ghristensen H, et al. Homocysteine and risk of recurrent stoke [J]. Stroke. 2003, 34: 1258-1261
[39] Buccianti G, Baragetti I, Bamonti F, et al. Plasma homocysteine levels and cardiovascular mortality in patients with end-stage renal disease [J]. J Nephrol. 2004, 17: 405-410
[40] Melfarb J, Menamin E, Monagle E. Plasma aminothiol oxidation in chronic hemodialysis patients[J]. Kidney International. 2002, 61(2): 705-716
[41] Wang R, Tang AG. Simultaneous Determination of Kynurenine and Tryptophan in Serum by High Performance Liquid Chromatography [J]. Chin J Chromatogr. 2006, 24: 140-143
[42] Xiao LD, Lou XB, Pi LG, et al. Simultaneous determination of kynurenine and kynurenic acid concentrations in human serum by HPLC with dual wavelengthsfluorescence detection [J]. Clin Chim Acta. 2008, 395: 178-180
[43] Eriksson L, Johasson E, Kettaneh W. et al. Multi-and Megavariate Date Analysis: Principles and applications [J]. Umetries Academy. 2001, 123-131.
[44] Lutz U, Lutz R W, Lutz W K. Metabolic Profiling of Glucuronides in Human Uring by LC-MS/MS and Partial Least-Squares Discriminant Analysis for Classition and Prediction of Gender. Analytical Chem. 2006, 78: 4567-4571.
[45] Pastore A, Massoud R, Motti C, et al. Fully Automated assay for total homocysteine, cysteine, cysteinylglycine, glutathione, cysteamine, and 2-mercaptopropionylglycine in plasma and urine[J]. Clinical Chemistry. 1998, 44: 825-832
[46] Chou S T, Yang CS, Ko L E. High performance liquid chromatography with fluorimetric detection for the determination of total homocysteine in human plasma: method and clinical applications [J]. Anal Chim Acta. 2001, 429: 331-336
[47] Kang S H, Kim J W, Chung D S, et al. Determination of homocysteine and other thiols in human plasma by capillary electrophoresis [J]. Biomed Anal. 1997, 15: 1435-1438
[48] Alberto J, Garcia, Rafael A C. Plasma total homocysteine quantification: an improvement of the classical high-performance liquid chromatographic method with fluorescence detection of the thiol-SBD derivatives [J]. Journal of Chromatography B. 2002, 779: 359-363
[49] Ueland PM, Refsum H, Stabler SP, et al. Total homocystine in plasma or serum methods and clinical applications [J]. Clin Chem. 1993, 39: 1746-1749
[50] Ramussan K, Moller J, Lyngbak M. Mithin-person variation of plasma homocysteine and effects of posture and tourniquet application [J]. Clinical Chemistry. 1999, 45(10): 1850-1855
[51] Andersson A, Lsaksson A, Hultberg B. Homocysteine export from erythrocytes and its implication for plasma sampling [J]. Clin Chem. 1992, 38: 1311-1315
[52] Ducros V, Candito M, Causse E, et al. Plasma homocysteine measurement: a study of preanalytical variation factors for conditions for total plasma homocysteineconcentrations [J]. Ann Biol Clin. 2001, 59: 33-39
[53] Theoret Y, Rivard G E, Infante Rivard C, et al. Stability of plasma homocysteine in perinatology [J]. Clinica Chimica Acta. 2002, 319: 63-66
[54] Moller J, Rasmussen K. Homocysteine determination [J]. Clin Chem Acta. 2001, 309: 53-56
[55] Miner S E S, Evroski J, Cole D E C. Theclinical chemistry and molecular biology of homocysteine metabolism: an update [J]. Clin Biochem. 1997, 30: 189-201.
[56] Diane M H, Lisa J J. Effect of temperature on stability of blood homocysteine in collection tubes containing 3-deazaadenosine [J]. Clinical Chemistry. 2002, 48(11): 2017-2022.
[57] Brandl R, Probst R, Muller B, et al. Evaluation of the measurement of homocysteine in patients with symptomatic arterial disease and in healthy volunteers[J]. Clin Chem. 1999, 45: 699-702.
[58] Arnadottir M , Berg AL , Hegbrant J , et al. Influence of haemodialysis on plasma total homocysteine concentration [J ] . Nephrol Dial Transplant. 1999, 14(1):142-145