高效液相色谱—荧光法同时测定重性抑郁障碍患者血清酪氨酸、5-羟色胺和色氨酸
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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。
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.
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。
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.
引文
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[63]皮兰敢,唐爱国,莫喜明,等.高效液相色谱-荧光法测定血清犬尿喹啉酸和色氨酸方法的建立.中华检验医学杂志,2007,30：1134-1137
[1]Kim CJ, Kovacs-Nolan JA, Yang C, et al.1-Tryptophan exhibits therapeutic function in a porcine model of dextran sodium sulfate (DSS)-induced colitis[J]. Nutr Biochem,2010,21(6):468-475.
[2]Gould GG, Hensler JG, Burke TF, et al. Density and function of central serotonin (5-HT) transporters,5-HT(1A) and 5-HT(2A) receptors, and effects of their targeting on BTBR T+tf/J mouse social behavior[J]. Neurochem,2011, 116(2):291-303.
[3]Bjork K, Sjogren B, Svenningsson P. Regulation of serotonin receptor function in the nervous System by lipid rafts and adaptor proteins. Exp Cell Res,2010, 316(8):1351-1356.
[4]Pallanti S, Bernardi S, Allen A, et al. Serotonin function in pathological gambling:blunted growth hormone response to Sumatriptan. Psychopharmacol,2010,24(12):1802-1809.
[5]Van Gool AR, Verkerk R, Fekkes D, et al. Neurotoxic and neuroprotective metabolites of kynurenine in patients with renal cell carcinoma treated with interferon-alpha:course and relationship with psychiatric status[J]. Psychiatry Clin Neurosci,2008,62(5):597-602.
[6]Chiarugi A, Calvani M, Meli E, et al. Synthesis and release of neurotoxic kynurenine metabolites by human monocyte-derived macrophages[J]. Neuroimmunol,2001,120(1-2):190-198.
[7]Fulop F, Szatmari I, Vamos E, et al. Syntheses, transformations and pharmaceutical applications of kynurenic acid derivatives. Curr Med Chem, 2009,16(36):4828-4842.
[8]Xiang ZY, Tang AG, Ren YP, et al. Simultaneous determination of serum tryptophan metabolites in patients with systemic lupus erythematosus by high performance liquid chromatography with fluorescence detection[J]. Clin Chem Lab Med,2010,48(4):513-517.
[9]Luo XB, Tang AG, Pi LG, et al. Determination of kynurenine in serum by high-performance liquid chromatography with on-column fluorescence derivatization[J]. Clin Chim Acta,2008,389(1-2):186-188.
[10]Pi LG, Tang AG, Mo XM, et al. More rapid and sensitive method for simultaneous determination of tryptophan and kynurenic acid by HPLC[J]. Clin Biochem,2009,42(4-5):420-425.
[11]Ishii Y, Ogara A, Katsumata T, et al. Quantification of nitrated tryptophan in proteins and tissues by high-performance liquid chromatography with electrospray ionization tandem mass spectrometry[J]. Pharm Biomed Anal,2007, 44(1):150-159.
[12]Cabordery AC, Toussaint M, Bonte JP,et al. Separation of Tic-hydantoin enantiomers, potent sigma-1 agonists, by high performance liquid chromatography and capillary electrophoresis[J]. Chromatogr A,2010, 1217(24):3871-3875.
[13]董存岩,皮敢兰,唐爱国,等.108例健康成人血清犬尿喹啉酸含量测定[J].中国现代医学杂志,2008,18(7)：920-921.
[14]Smith AJ, Smith RA, and Stone TW.5-Hydroxyanthranilic acid, a tryptophan metabolite, generates oxidative stress and neuronal death via p38 activation in cultured cerebellar granule neurones[J]. Neurotox Res,2009,15(4):303-310.
[15]Yao JK, Dougherty GG, Reddy RD, et al. Altered interactions of tryptophan metabolites in first-episode neuroleptic-naive patients with schizophrenia. Mol Psychiatry,2010,15(9):938-953.
[16]Uberos J, Romero J, Molina CA, et al. Melatonin and elimination of kynurenines in children with Down's syndrome[J]. Pediatr Endocrinol Metab,2010, 23(3):277-282.
[17]Forrest CM, Mackay GM, Stoy N, et al. Blood levels of kynurenines, interleukin-23 and soluble human leucocyte antigen-G at different stages of Huntington's disease[J]. Neurochem,2010,112(1):112-122.
[18]Potter MC, Elmer GI, Bergeron R, et al. Reduction of endogenous kynurenic acid formation enhances extracellular glutamate, hippocampal plasticity, and cognitive behavior. Neuropsychopharmacology,2010,35(8):1734-1742.
[19]Gulaj E, Pawlak K, Bien B, et al. Kynurenine and its metabolites in Alzheimer's disease patients[J]. Adv Med Sci,2010,55(2):204-211.
[20]Kim YK, Myint AM, Verkerk R, et al. Cytokine changes and tryptophan metabolites in medication-naive and medication-free schizophrenic patients [J]. Neuropsychobiology,2009,59(2):123-129.
[21]Mysliwiec P, Mysliwiec H, Pawlak D, et al. Tryptophan and its metabolites in renal allograft recipients[J]. Przegl Lek,2009,66(3):115-118.
[22]Pawlak K, Mysliwiec M and Pawlak D. Hypercoagulability is independently associated with kynurenine pathway activation in dialysed uraemic patients [J]. Thromb Haemost,2009,102(1):49-55.
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[1]Kim CJ, Kovacs-Nolan JA, Yang C, et al.1-Tryptophan exhibits therapeutic function in a porcine model of dextran sodium sulfate (DSS)-induced colitis[J]. Nutr Biochem,2010,21(6):468-475.
[2]Gould GG, Hensler JG, Burke TF, et al. Density and function of central serotonin (5-HT) transporters,5-HT(1A) and 5-HT(2A) receptors, and effects of their targeting on BTBR T+tf/J mouse social behavior[J]. Neurochem,2011, 116(2):291-303.
[3]Bjork K, Sjogren B, Svenningsson P. Regulation of serotonin receptor function in the nervous System by lipid rafts and adaptor proteins. Exp Cell Res,2010, 316(8):1351-1356.
[4]Pallanti S, Bernardi S, Allen A, et al. Serotonin function in pathological gambling:blunted growth hormone response to Sumatriptan. Psychopharmacol,2010,24(12):1802-1809.
[5]Van Gool AR, Verkerk R, Fekkes D, et al. Neurotoxic and neuroprotective metabolites of kynurenine in patients with renal cell carcinoma treated with interferon-alpha:course and relationship with psychiatric status[J]. Psychiatry Clin Neurosci,2008,62(5):597-602.
[6]Chiarugi A, Calvani M, Meli E, et al. Synthesis and release of neurotoxic kynurenine metabolites by human monocyte-derived macrophages[J]. Neuroimmunol,2001,120(1-2):190-198.
[7]Fulop F, Szatmari I, Vamos E, et al. Syntheses, transformations and pharmaceutical applications of kynurenic acid derivatives. Curr Med Chem, 2009,16(36):4828-4842.
[8]Xiang ZY, Tang AG, Ren YP, et al. Simultaneous determination of serum tryptophan metabolites in patients with systemic lupus erythematosus by high performance liquid chromatography with fluorescence detection[J]. Clin Chem Lab Med,2010,48(4):513-517.
[9]Luo XB, Tang AG, Pi LG, et al. Determination of kynurenine in serum by high-performance liquid chromatography with on-column fluorescence derivatization[J]. Clin Chim Acta,2008,389(1-2):186-188.
[10]Pi LG, Tang AG, Mo XM, et al. More rapid and sensitive method for simultaneous determination of tryptophan and kynurenic acid by HPLC[J]. Clin Biochem,2009,42(4-5):420-425.
[11]Ishii Y, Ogara A, Katsumata T, et al. Quantification of nitrated tryptophan in proteins and tissues by high-performance liquid chromatography with electrospray ionization tandem mass spectrometry[J]. Pharm Biomed Anal,2007, 44(1):150-159.
[12]Cabordery AC, Toussaint M, Bonte JP,et al. Separation of Tic-hydantoin enantiomers, potent sigma-1 agonists, by high performance liquid chromatography and capillary electrophoresis[J]. Chromatogr A,2010, 1217(24):3871-3875.
[13]董存岩,皮敢兰,唐爱国,等.108例健康成人血清犬尿喹啉酸含量测定[J].中国现代医学杂志,2008,18(7)：920-921.
[14]Smith AJ, Smith RA, and Stone TW.5-Hydroxyanthranilic acid, a tryptophan metabolite, generates oxidative stress and neuronal death via p38 activation in cultured cerebellar granule neurones[J]. Neurotox Res,2009,15(4):303-310.
[15]Yao JK, Dougherty GG, Reddy RD, et al. Altered interactions of tryptophan metabolites in first-episode neuroleptic-naive patients with schizophrenia. Mol Psychiatry,2010,15(9):938-953.
[16]Uberos J, Romero J, Molina CA, et al. Melatonin and elimination of kynurenines in children with Down's syndrome[J]. Pediatr Endocrinol Metab,2010, 23(3):277-282.
[17]Forrest CM, Mackay GM, Stoy N, et al. Blood levels of kynurenines, interleukin-23 and soluble human leucocyte antigen-G at different stages of Huntington's disease[J]. Neurochem,2010,112(1):112-122.
[18]Potter MC, Elmer GI, Bergeron R, et al. Reduction of endogenous kynurenic acid formation enhances extracellular glutamate, hippocampal plasticity, and cognitive behavior. Neuropsychopharmacology,2010,35(8):1734-1742.
[19]Gulaj E, Pawlak K, Bien B, et al. Kynurenine and its metabolites in Alzheimer's disease patients[J]. Adv Med Sci,2010,55(2):204-211.
[20]Kim YK, Myint AM, Verkerk R, et al. Cytokine changes and tryptophan metabolites in medication-naive and medication-free schizophrenic patients [J]. Neuropsychobiology,2009,59(2):123-129.
[21]Mysliwiec P, Mysliwiec H, Pawlak D, et al. Tryptophan and its metabolites in renal allograft recipients[J]. Przegl Lek,2009,66(3):115-118.
[22]Pawlak K, Mysliwiec M and Pawlak D. Hypercoagulability is independently associated with kynurenine pathway activation in dialysed uraemic patients [J]. Thromb Haemost,2009,102(1):49-55.
[23]Schefold JC, Zeden JP, Fotopoulou C, et al. Increased indoleamine 2,3-dioxygenase (IDO) activity and elevated serum levels of tryptophan catabolites in patients with chronic kidney disease:a possible link between chronic inflammation and uraemic symptoms. Nephrol Dial Transplant,2009, 24(6):1901-1908.
[24]Pawlak K, Kowalewska A, Mysliwiec M, et al. Kynurenine and its metabolites-kynurenic acid and anthranilic acid are associated with soluble endothelial adhesion molecules and oxidative status in patients with chronic kidney disease[J]. Am J Med Sci,2009,338(4):293-300.
[25]Pawlak D, Tankiewicz A, Matys T, et al. Peripheral distribution of kynurenine metabolites and activity of kynurenine pathway enzymes in renal failure [J]. Physiol Pharmacol,2003,54(2):175-189.
[26]Narui K, Noguchi N, Saito A, et al. Anti-infectious activity of tryptophan metabolites in the L-tryptophan-L-kynurenine pathway[J]. Biol Pharm Bull, 2009,32(1):41-44.
[27]Opitz CA, Wick W, Steinman L, et al. Tryptophan degradation in autoimmune diseases. Cell Mol Life Sci,2007,64(19-20):2542-2563.
[28]Schroecksnadel K, Winkler C, Duftner C, et al. Tryptophan degradation increases with stage in patients with rheumatoid arthritis[J]. Clin Rheumatol, 2006,25(3):334-337.
[29]Maneglier B, Malleret B, Guillemin GJ, et al. Modulation of indoleamine-2,3-dioxygenase expression and activity by HIV-1 in human macrophages[J]. Fundam Clin Pharmacol,2009,23(5):573-581.
[30]Cozzi A, Zignego AL, Carpendo R, et al. Low serum tryptophan levels, reduced macrophage IDO activity and high frequency of psychopathology in HCV patients[J]. Viral Hepat,2006,13(6):402-408.
[31]Schrocksnadel K, Widner B, Bergant A, et al. Tryptophan degradation during and after gestation[J]. Adv Exp Med Biol,2003,527:77-83.
[32]DiNatale BC, Murray IA, Schroeder JC, et al. Kynurenic acid is a potent endogenous aryl hydrocarbon receptor ligand that synergistically induces interleukin-6 in the presence of inflammatory signaling[J]. Toxicol Sci,2010, 115(1):89-97.
[33]Schefold JC, Zeden JP, Pschowski R, et al. Treatment with granulocyte-macrophage colony-stimulating factor is associated with reduced indoleamine 2,3-dioxygenase activity and kynurenine pathway catabolites in patients with severe sepsis and septic shock[J]. Scand J Infect Dis,2010, 42(3):164-171.
[34]Serbecic N, Lahdou I, Scheuerle A, et al. Function of the tryptophan metabolite, L-kynurenine, in human corneal endothelial cells[J]. Mol Vis,2009, 15:1312-1324.