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目的 :分析糖尿病患者与正常对照组微量血清中胆汁酸各组分水平的差异,并探讨与血糖的关系。方法 :建立测定微量血清中9种胆汁酸的超高效液相色谱-串联质谱(UHPLC-MS/MS)方法。选取15例糖尿病患者和10名正常对照者作为研究对象。血清样本经乙腈沉淀蛋白,采用Waters Atlantis T3柱分离,以甲醇-水(含乙酸)为流动相,用负离子多反应监测模式分析,检测空腹血清胆汁酸浓度。并且分析血清胆汁酸水平在2组间的差异及其与血糖和胰岛素的关系。结果:正常人体血清中以游离型胆汁酸和甘氨结合型胆汁酸为主。相比于健康对照组,糖尿病患者血清中甘氨鹅脱氧胆酸[7.24(3.34,10.83)mg/L比15.71(11.39,27.12)mg/L,P<0.05]和甘氨胆酸[1.41(0.96,1.88)mg/L比2.38(1.50,3.45)mg/L,P<0.05]显著升高,而2组甘氨脱氧胆酸、鹅脱氧胆酸、脱氧胆酸、胆酸无显著性差异。空腹血糖与甘氨鹅脱氧胆酸及甘氨胆酸水平呈正相关(r=0.470,r=0.405,均P<0.05)。结论:建立了一种在微量血清中同时检测9种胆汁酸的灵敏、有效的UHPLC-MS/MS方法,为深入研究胆汁酸在疾病中的作用提供了一定的基础。利用该方法初步发现甘氨酸结合型胆汁酸(甘氨鹅脱氧胆酸和甘氨胆酸)水平在糖尿病患者血清中比例增加,提示胆汁酸组分的改变可能与糖代谢异常有关。
OBJECTIVE: To analyze the difference of bile acid components in patients with diabetes mellitus and normal controls, and to explore the relationship with bile acids. Methods: To establish a method for the determination of nine bile acids in micro-serum by ultra performance liquid chromatography-tandem mass spectrometry (UHPLC-MS / MS). Fifteen diabetic patients and 10 normal controls were selected as research subjects. Serum samples were precipitated with acetonitrile, separated on a Waters Atlantis T3 column, and analyzed for fasting serum bile acid concentrations using methanol-water (containing acetic acid) as a mobile phase by negative ion multiple reaction monitoring. And analyze the difference of serum bile acid level between two groups and its relationship with blood glucose and insulin. Results: In normal human serum, free bile acid and glycine-bound bile acid were the main components. Compared with the healthy control group, glycochenodeoxycholic acid [7.24 (3.34,10.83) mg / L vs 15.71 (11.39, 27.12) mg / L, P <0.05] and glycocholic acid [1.41 0.96,1.88) mg / L than 2.38 (1.50,3.45) mg / L, P <0.05], but there was no significant difference between the two groups of glycodeoxycholic acid, chenodeoxycholic acid, deoxycholic acid and cholic acid . Fasting plasma glucose was positively correlated with glycochenodeoxycholic acid and glycocholic acid (r = 0.470, r = 0.405, all P <0.05). Conclusion: A sensitive and effective UHPLC-MS / MS method for simultaneous detection of nine bile acids in trace serum has been established, which provides a basis for further study on the role of bile acid in the disease. Using this method, it was found that the level of glycine-bound bile acids (glycitehenodeoxycholic acid and glycocholic acid) increased in the serum of diabetic patients, suggesting that the changes of bile acid components may be related to abnormal glucose metabolism.