血清支链和芳香族氨基酸同位素稀释质谱检测法的建立和应用研究  

Research on the Establishment and Application of Isotope Dilution Tandem Mass Spectrometry Method for Serum Branched-chain and Aromatic Amino Acids

作　　者：杨睿悦[1] 李红霞[1] 国汉邦[1] 赵海建[2] 王抒[1] 董军[1] 陈文祥[1,2] 

机构地区：[1]卫生部北京医院卫生部北京老年医学研究所,卫生部老年医学重点实验室,北京100730 [2]卫生部北京医院卫生部临床检验中心,北京100730

出　　处：《现代检验医学杂志》2013年第5期10-13,共4页Journal of Modern Laboratory Medicine

基　　金：国家自然科学基金项目（项目编号：30872413,81171647）.

摘　　要：目的 建立同位素稀释液相色谱串联质谱（ID-LC/MS/MS）同时测定血清五种支链和芳香族氨基酸（即缬氨酸、异亮氨酸、亮氨酸、酪氨酸和苯丙氨酸）的方法,分析其与糖尿病及心血管病危险因素的关系.方法 采用同位素标记被测物作内标,沉淀蛋白后LC/MS/MS分离检测,标准曲线定量;对不同水平血清样本进行五次重复分析,每次每种血清重复分析3份,以考察方法的精密度;测定血清样本添加不同浓度标准品的加样回收率.收集2010年10～12月111例健康志愿者空腹静脉血,离心分离血清,测定血清支链和芳香族氨基酸及传统危险因素指标水平,进行相关性分析.结果 五种氨基酸在该试验条件下分离良好,分析时间约4 min;测定不同血清水平的批内变异系数（CV）为1.56%（1.07%～2.02%）,总CV为2.03%（1.14%～3.01%）;多次试验的加样回收率范围为98.34%～103.25%.测定111例健康人血清支链和芳香族氨基酸水平与体重指数、空腹血糖、三酰甘油、高密度脂蛋白胆固醇酯化速率、肌酐、尿酸、载脂蛋白CII和CIII以及超敏C反应蛋白显著正相关（t=0.000 02～0.046,P值均〈0.05）;与高密度脂蛋白胆固醇显著负相关（t=0.000 5～0.048,P值均〈0.05）.结论 该研究建立了简便精密的ID-LC/MS/MS测定血清支链和芳香族氨基酸的方法,有望在糖尿病和心血管病危险分析中发挥作用.Objective To develop a method for measuring serum branched-chain and aromatic amino acids （i. e. valine,isoleu-cine,leucine,tyrosine and phenylalanine） by isotope dilution-liquid chromatography tandem mass spectrometry （ID-LC/MS/ MS） and analyze their correlation with risk factors of diabetes and cardiovascular diseases. Methods By using the isotope la-beled internal standard for each amino acid,after protein precipitation, serum samples were determined by LC/MS/MS and quantitated by the method of standard curve. To estimate the precision of the method,different frozen serum pools were re-peatedly analyzed in five runs,and every pool was analyzed in triplicate. In addition,the recovery rates were analyzed with the serum sample added with different levels of standard. Blood samples were collected from 111 apparently healthy subjects from October to December in 2010 and centrifuged,and the sera were separated. The levels of serum branched-chain and aro-matic amino acids were measured to investigate their correlation with traditional risk factors. Results The five amino acids were separated well,and the total analysis time was about 4.0 min. The within-run and total coefficients of variation （CV） were 1.56% （1.07%-2.02%） and 2.03% （1.14%-3.01%） ,respectively. The analytical recoveries ranged from 98.34% to 103.25 %. By the current method,serum concentrations of certain amino acids were determined in 111 healthy subjects. BCAAs and AAAs were positively correlated with BMI,Glucose,TG, fractional esterification rate of HDL-C, Creatinine, U-ric acid,ApoCIl,ApoCⅢ and hs-CRP （t=0. 000 02-0. 046,P〈0.05） ,and negatively correlated with HDL-C （t=0. 000 5 %0. 048,P〈0.05）. Conclusion An ID-LC/MS/MS method for serum BCAAs and AAAs has been established. The method is simple and precise,and may serve as a potentially useful technique in risk assessment of diabetes and cardiovascular disea-ses.

关 键 词：支链氨基酸 芳香族氨基酸 串联质谱法 糖尿病 危险因素 

分 类 号：Q503[生物学—生物化学]