尿素酶预处理-气相色谱-质谱法在甲基丙二酸尿症诊断中的应用  

Application of Urease Pretreatment-Gas Chromatography-Mass Spectrometry in the Diagnosis of Methylmalnic Aciduria

作　　者：郝虎[1] 宋元宗[2] 肖昕[1] 张春花 王自能[2] 

机构地区：[1]中山大学附属第六医院围产医学中心遗传代谢病实验室,广州510655 [2]暨南大学附属第一医院围产医学中心,广州510632 [3]日本松本生命科学研究所,日本金泽921-8154

出　　处：《实用儿科临床杂志》2008年第8期574-576,共3页Journal of Applied Clinical Pediatrics

基　　金：国家“973”项目资助(2001CB5103);广东省科技计划项目资助(2004B50301008)

摘　　要：目的探讨尿素酶预处理-气相色谱-质谱法（UP-GC-MS）在甲基丙二酸尿症（MMA）诊断、治疗中的价值。方法留取8例MMA患儿和15例同年龄健康儿童晨尿2mL,尿液标本经尿素酶去尿素、加内标正十七酸、除蛋白、真空干燥处理,残余物用双（三甲基硅烷基）三氟乙酰胺/三甲基氯硅烷（BSTFA/TMCS）衍生后进样,分析仪器为Finnigan GC-MS联用仪,每次取样0.5-1.0μL,扫描模式为全扫描加选择离子扫描模式,分流比10∶1,起始温度60℃,然后以17℃/min的速度升温至320℃。质谱电离方式为电子解离模式,扫描范围为m/z50-m/z650。应用SPSS12.0软件进行t检验。结果患儿尿液中可检测到明显的甲基丙二酸、甲基枸橼酸等MMA患儿尿液标志物信号。在总离子流图上,其绝对保留时间（相对保留时间）分别是6.78min（0.396min）和11.53min（0.24min）。在质谱图上,甲基丙二酸的特征离子（三甲基硅烷化后质荷比,TMSm/z）分别为73、147、247。8例MMA患儿尿液甲基丙二酸水平为1.72-18.2nmol/mgCr;健康对照组15例儿童为0.001-0.202nmol/mgCr。经过lg（x＋5）对数转换后（其中x为原始数据）,病例组尿液甲基丙二酸水平为（0.703±0.005）,健康对照组为（0.973±0.184）,二组比较差异具有统计学意义（t=4.10P〈0.01）。结论建立UP-GC-MS分析尿液代谢成分超越了有机酸萃取法的一些局限,是MMA确诊的重要方法。Objective To explore the value of urease pretreatment - gas chromatography - mass spectrometry （ UP - GC - MS） in the diagnosis and therapy of methylmalonic aciduria （MMA）. Methods Urine of 8 children with MMA and 15 nolznal children （ with the same age） were collected. Urine samples were decomposed with urease,heptadecanoie acid was added as an internal standard,protein was denaturized with ethanol and precipitate was removed by centrifugation, evaporation was pelformed to dryness, the residue was trimethylsilylly derivati- zed with his （trimethylsilyl） trifluoroacetamide/trimethyl -chlorosilane （BSTFA/TMCS）. The instrument was Finnigan GC - MS. Aliquots of 0.5 - 1.0 μL was used for each sample. The scan mode was full scan and selective ion monitoring pattern. Each sample was injected in 10 ： 1 split mode. The temperature was raised from 60 ℃ to 320 ℃ with 17 ℃/rain. Mass spectrum was scanned by electron impact mode from m/z 50 to m/z 650, and then the results were analyzed with SPSS 12.0 software. Results Large quantity of methylmalonic acid and methylcitrate were detected in the urine sample. In total ion current profile, their retention time （ relative retention time） were 6.78 min （0. 396 min） , 11.53 min （0.24 min） respectively,with characteristic ions of MMA in mass spectrums （ mass chromatograms of trimethylsilyl, TMS m/z） were 73, 147 and 247 respectively. The level of methylmalonic acid in urine of MMA group was 1.72 - 18.2 nmol/mg creatinine, and the control group was 0.001 -0.202 nmol/mg cre atinine. The data were transfered by lg （x ＋ 5 ）, the level of MMA group was （0. 703 ±0. 005 ） and the control group was （ 0. 973 ± 0.184 ）, the level of methylmalonic acid in MMA group was significantly higher than that in control group （ t = 4. 10 P 〈 0.01 ）. Conclusion Establishment of the approach UP - GC - MS to analyze urinary metabolites beyond the limitation of organic acids is a reliable method for the diagnosis of MMA.

关 键 词：遗传代谢病 甲基丙二酸尿症 尿素酶预处理-气相色谱-质谱法 

分 类 号：R725.8[医药卫生—儿科]