NMR联用HILIC-UPLC-MS/MS测定TNBS诱导的结肠炎大鼠尿液和血浆代谢物变化

Combined NMR and HILIC-UPLC-MS/MS Analysis to Detect Metabolic Changes of Urine and Plasma from Rats with TNBS-induced Colitis

【目的】基于非靶标和靶标代谢组学技术,探讨三硝基苯磺酸(TNBS)诱导的结肠炎模型大鼠尿液以及血浆中内源性代谢物的变化。【方法】16只雄性SD大鼠随机分成对照组和模型组,每组8只,对模型组大鼠给予TNBS灌肠以建立实验性大鼠结肠炎模型。利用核磁共振氢谱(^(1)H NMR)检测两组大鼠尿液代谢物,同时联用HILIC模式超高效液相色谱串联三重四级杆质谱(HILIC-UPLC-MS/MS)测定以上大鼠尿液以及血浆中氨基酸水平,并通过多元变量统计方法对代谢组学数据进行分析。【结果】同对照组相比,^(1)H NMR结果表明模型组大鼠尿液代谢轮廓发生明显改变,其中丙酮酸、甲酸、甲胺与柠檬酸水平升高,氧化三甲胺和丙二酸水平降低(均P<0.05);HILICUPLC-MS/MS结果显示模型组大鼠尿液中苯丙氨酸、组氨酸水平显著上升,血浆中赖氨酸、精氨酸、苯丙氨酸、亮氨酸、甘氨酸、色氨酸、脯氨酸、组氨酸和酪氨酸水平显著升高,但谷氨酰胺、缬氨酸、丙氨酸和异亮氨酸水平明显降低(均P<0.05)。【结论】经TNBS诱导的结肠炎模型大鼠,其能量代谢、胺类代谢以及氨基酸代谢通路受到影响;采用的1H NMR与HILIC-UPLC-MS/MS联用的多元代谢组学方法,揭示了结肠炎大鼠尿液及血浆代谢物的变化,为炎症性肠病的机制研究与相关生物标志物的探索提供了新的思路。

【Objective】The aim of this study was to investigate the variations of endogenous metabolites in the urine and plasma of 2,4,6-trinitro benzene sulfonic acid(TNBS)-induced colitis model rats on the base of untargeted and targeted metabolomics techniques.【Methods】A total of 16 male SD rats were randomly and equally divided into control group and model group.The rats in the model group were treated with TNBS enema to establish experimental colitis rat model.The urinary metabolites of rats in the both two groups were detected by employing ^(1)H nuclear magnetic resonance(^(1)H NMR).The levels of amino acids in urine and plasma of above rats were investigated by hydrophilic interaction ultra-highperformance liquid chromatography-tandem mass spectrometry(HILIC-UPLC-MS/MS).Multivariate statistical analysis was combined to analyze the metabolic profile additionally.【Results】Compared with the control group,the 1H NMR results showed that the metabolic profile of urine in the model rats was significantly changed,including increased levels of pyruvate,formate,methylamine and citrate,decreased levels of trimethylamine oxide and malonate(all P<0.05).Meanwhile,the HILIC-UPLC-MS/MS results indicated that the levels of phenylalanine and histidine were significantly increased in the urine of model rats,the contents of lysine,arginine,phenylalanine,leucine,glycine,tryptophan,proline,histidine and tyrosine were markedly elevated while glutamine,valine,alanine and isoleucine were notably reduced in the plasma of model rats(all P<0.05).【Conclusions】The metabolic pathways including energy metabolism,amine metabolism and amino acid metabolism were interfered in colitis rats induced by TNBS.The multivariate metabolomics methods based on1H NMR and HILIC-UPLC-MS/MS revealed the metabolic alterations of urine and plasma in the colitis rats,providing new perspective for the mechanism research of inflammatory bowel disease(IBD)and the exploration of related biomarkers.