超高效液相色谱-串联质谱法检测人微量血清中9种胆汁酸及其临床意义

Determination of nine bile acids in micro-amount of serum by ultra-high performance liquid chromatographytandem mass spectrometry and its clinical significance

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 investigate the differences of serum levels of bile acids between patients with type 2 diabetes and healthy controls, and analyze the relationship between serum levels of bile acids and plasma glucose. Methods A sensitive method was established based on ultra-high performance liquid chromatography-tandem mass spectrometry（UHPLC-MS/MS） for analyzing nine bile acids in micro-amount of serum. Serum samples from 15 type 2 diabetes patients and 10 healthy controls were tested and analyzed. Protein precipitation with acetonitrile was used for sample preparation.The analytes were eluted using methanol-water buffer（containing acetic acid） as mobile phase on a Waters Atlantis T3 column. Then MS analysis of bile acids was performed by multiple reaction monitoring mode, and the fasting serum levels of bile acids were detected. Furthermore, the difference in levels of bile acids between the two groups and the associations between levels of bile acids and glucose as well as insulin were also analyzed. Results In healthy subjects, free bile acids and glycine conjugated bile acids were the bile acids with the highest level. Glycochenodeoxycholic acid（GCDCA） [7.24（3.34, 10.83） mg/L vs 15.71（11.39, 27.12） mg/L, P〈0.05] and glycocholic acid（GCA） [1.41（0.96, 1.88） mg/L vs 2.38（1.50, 3.45） mg/L, P〈0.05] levels were significantly higher in patients with type 2 diabetes than in healthy controls.However, no significant differences in fasting serum levels of glycodeoxycholic acid, chenodeoxycholic acid, deoxycholic acid and cholic acid were observed between diabetic subjects and healthy controls. Analysis demonstrated the positive association of fasting plasma glucose with serum GCDCA（r=0.470, P〈0.05） and GCA（r=0.405, P〈0.05）. Conclusions UHPLC-MS/MS can efficiently differentiate and quantify nine bile acids in micro-amount of serum and provide the basis for studying the effects of bile acids in the disease. Our preliminary results showed that levels of serum glycine conjugated bile acids（GCDCA and GCA） were increased in diabetic subjects, suggesting the alteration in bile acids composition may associate with glucose in tolerance.