HPLC同时测定关节炎大鼠血清色氨酸和犬尿氨酸
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摘要
建立新的高效液相色谱法(HPLC),检测佐剂性关节炎(AA)大鼠血清中色氨酸(Trp)和犬尿氨酸(Kyn)。SD大鼠成模后取血清,用蛋白沉淀法处理,离心后取上清液进样。考察该方法的专属性、标准曲线、精密度、回收率和稳定性等能否用于AA大鼠Trp和Kyn含量测定。Trp保留时间9.0 min,线性范围25～1 000μmol/L,检测限0.27μmol/L;Kyn保留时间5.6 min,线性范围0.5～10μmol/L,检测限0.07μmol/L;平均回收率在80%～120%范围之内;日内、日间精密度RSD均小于15%,符合生物样品定量分析方法要求,并可应用于AA大鼠血清中Trp和Kyn测定。建立了一种灵敏度高、快速的HPLC分析方法,适于测定AA大鼠血清中Trp和Kyn含量。
To establish a high performance liquid chromatography(HPLC) method for determination tryptophan(Trp) and kynurenine(Kyn) in serum from rats with adjuvant induced arthritis(AA). A model was established in SD rats, and the rat serum was taken after model formation and treated with protein precipitation, after centrifugation, the supernatant was taken for injection. The specificity, standard curve, precision, recovery and stability of the method were investigated could be used for the determination of Trp and Kyn in AA rats. The retention time of Trp was about 9.0 min, the linear range was 25～1 000 μmol/L, the detection limit was 0.27 μmol/L, the retention time of Kyn was about 5.6 min, the linear range was 0.5～10 μmol/L, and the detection limit was 0.07 μmol/L. The average recovery rate is in the range of 80%～120%; the intra-day and inter-day precision RSD were less than 15%. These were in line with the requirements of the biological sample quantitative analysis method, and could be applied to the quantitative determination of Trp and Kyn in serum from rats with AA. The HPLC method with high sensitivity and rapidity is established,and suitable for the determination of Trp and Kyn in rat serum.
To establish a high performance liquid chromatography(HPLC) method for determination tryptophan(Trp) and kynurenine(Kyn) in serum from rats with adjuvant induced arthritis(AA). A model was established in SD rats, and the rat serum was taken after model formation and treated with protein precipitation, after centrifugation, the supernatant was taken for injection. The specificity, standard curve, precision, recovery and stability of the method were investigated could be used for the determination of Trp and Kyn in AA rats. The retention time of Trp was about 9.0 min, the linear range was 25～1 000 μmol/L, the detection limit was 0.27 μmol/L, the retention time of Kyn was about 5.6 min, the linear range was 0.5～10 μmol/L, and the detection limit was 0.07 μmol/L. The average recovery rate is in the range of 80%～120%; the intra-day and inter-day precision RSD were less than 15%. These were in line with the requirements of the biological sample quantitative analysis method, and could be applied to the quantitative determination of Trp and Kyn in serum from rats with AA. The HPLC method with high sensitivity and rapidity is established,and suitable for the determination of Trp and Kyn in rat serum.
引文
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[13] Zhao J,Chen H,Ni P,et al.Simultaneous determination of urinary tryptophan,tryptophan-related metabolites and creatinine by high performance liquid chromatography with ultraviolet and fluorimetric detection[J].J Chromatogr B Analyt Technol Biomed Life Sci,2011,879(26):2720-5.
[14] Owe-Young R,Webster N L,Mukhtar M,et al.Kynurenine pathway metabolism in human blood-barrier cells:implications for immune tolerance and neurotoxicity[J].J Neurochem,2008,105:1346-57.
[2] D′Amato N C,Rogers T J,Gordon M A,et al.A TDO2-AhR signaling axis facilitates anoikis resistance and metastasis in triple-negative breast cancer[J].Cancer Res,2015,75(21):4651-64.
[3] Lanz T V,Williams S K,Stojic A,et al.Tryptophan-2,3-Dioxygenase(TDO) deficiency is associated with subclinical neuroprotection in a mouse model of multiple sclerosis[J].Sci Rep,2017,7:41271.
[4] Cervenka I,Agudelo L Z,Ruas J L.Kynurenines:Tryptophan′s metabolites in exercise,inflammation,and mental health[J].Science,2017,357(6349):1-8.
[5] 宋珊珊,张玲玲,魏伟.实验性关节炎动物模型建立及病理机制研究进展[J].中国药理学通报,2011,27(12):1648-52.
[6] Zhao J.Simultaneous determination of plasma creatinine,uric acid,kynurenine and kynurenine by high-performance liquid chromatography:method validation and inapplication to the assessment of renal function[J].Biomed Chromatogr,2015,29(3):410-5.
[7] Iizuka H,Hirasa Y,Kubo K,et al.Enantiomeric separation of D,L-tryptophan and D,L-kynurenine by HPLC using pre-column fluorescence derivatization with R(-)-DBD-PyNCS[J].Biomed Chromatogr,2011,25(7):743-7.
[8] Chang Y,Wu Y,Wei W,et al.Therapeutic effects of TACI-Ig on rats with adjuvant-induced arthritis via attenuating inflammatory responses[J].Rheumatology(Oxford),2011,50(5):862-70.
[9] Chen Y,Guillemin G J.Kynurenine pathway metabolites in humans:disease and healthy states[J].Int J Tryptophan Res,2009,2:1-19.
[10] Badawy A A.Tryptophan availability for kynurenine pathway metabolism across the life span:Control mechanisms and focus on aging,exercise,diet and nutritional supplements[J].Neuropharmacology,2017,112(Pt B):248-63.
[11] Pertovaara M,Raitala A,Uusitalo H,et al.Mechanisms dependent on tryptophan catabolism regulate immune responses in primary Sj?gren′s syndrome[J].Clin Exp Immunol,2005,142(1):155-61.
[12] Jeffrey K,Benjamin S,Robert H W,et al.Extraction and quantification of tryptophan and kynurenine from cultured cells and media using a high performance liquid chromatography(HPLC) system equipped with an ultrasensitive diode array detector[J].Bio Protoc,2016,6(7):e1781.
[13] Zhao J,Chen H,Ni P,et al.Simultaneous determination of urinary tryptophan,tryptophan-related metabolites and creatinine by high performance liquid chromatography with ultraviolet and fluorimetric detection[J].J Chromatogr B Analyt Technol Biomed Life Sci,2011,879(26):2720-5.
[14] Owe-Young R,Webster N L,Mukhtar M,et al.Kynurenine pathway metabolism in human blood-barrier cells:implications for immune tolerance and neurotoxicity[J].J Neurochem,2008,105:1346-57.