尿滤纸片法气相色谱2质谱分析技术

・遗传代谢内分泌疾病研究・基金项目:国家自然科学基金委员会国家杰出青年科学基金(30125019);卫生部临床学科重点建设项目(97070240)作者单位:430030武汉,华中科技大学同济医学院附属同济医院儿科,同济医学院遗传代谢性疾病诊断中心尿滤纸片法气相色谱2质谱分析技术在遗传性代谢病高危筛查诊断中的应用罗小平　王慕逖　魏虹　梁雁　王宏伟　林汉华　董永绥　刘皖君　方俊敏　宁琴 【摘要】　目的　建立一种适合我国国情的有机酸尿症及其他遗传性代谢病的高危筛查诊断方法。

方法　应用滤纸片收集尿标本,建立尿有机酸及氨基酸代谢产物的萃取、衍化和气相色谱2质谱分析方法;成立遗传代谢性疾病高危筛查诊断协作网络并开展工作。

结果　352例样本中共检出阳性病例34例(9166%),病种包括甲基丙二酸血症6例,α2酮戊二酸尿症5例,酪氨酸血症Ⅰ型4例,二羧酸尿症4例,多种羧化酶缺乏症3例,苯丙酮尿症3例,高乳酸血症3例,丙酸血症2例,鸟氨酸氨甲酰基转移酶缺乏症1例,乙基丙二酸2己二酸尿症1例,戊二酸尿症Ⅱ型1例和32甲基巴豆酰羧化酶缺乏症1例。

常见的临床症状和体征包括智能发育落后、惊厥、肌张力异常和黄疸。

常规实验室检查多见代谢性酸中毒、低血糖和高血氨等异常。

结论　尿有机酸气相色谱2质谱分析为遗传性代谢病高危筛查诊断的重要技术。

应用滤纸片收集尿标本后在遗传代谢病诊断中心进行检测为实用而有效的方法。

建立协作网络是成功开展遗传性代谢病高危筛查诊断工作的关键。

【关键词】　代谢缺陷,先天性;　有机酸类;　碎片质谱法;　尿分析Application of gas chrom atography 2m ass spectrometry analysis on urine filter p aper in the high 2risk screening and diagnosis of inherited metabolic diseases LUO Xiao 2ping ,WANG Mu 2ti ,WEI Hong ,LIANG Yan ,WANG Hong 2wei ,LIN Han 2hua ,DONG Yong 2sui ,LIU Wan 2jun ,F ANG Jun 2min ,NING Qin 1Department o f Pediatrics ,Tongji Hospital ,Center for the Diagnosis o f G enetic Metabolic Diseases ,Tongji Medical College ,Huazhong Univer sity o f Science and Technology ,Wuhan 430030,China【Abstract 】　Objective T o establish a specific procedure for the high 2risk screening and diagnosis of organic acidurias and other inherited metabolic diseases in China 1Methods A nation 2wide netw ork for the high 2risk screening and diagnosis of genetic metabolic diseases was formed to facilitate the collaboration 1Urine samples were collected using filter paper from patients with clinical sym ptoms suspicious of inherited metabolic diseases 1The samples were eluted with distilled water and internal standards were added 1Samples were treated with hydroxylamine hydrochloride to form oximes to im prove the recoveries of 22ketoacids 1Urinary organic acids were extracted with ethyl acetate and diethyl ether under acidic condition 1A fter dehydration ,the combined organic phase was evaporated to dryness with nitrogen 1The residues were added with BSTFA +1%T MCS and heat incubated to form the trimethylsilyl derivatives ,and then were analyzed on an Agilent 5890/5973N gas chromatography 2mass spectrometer (G C 2MS ),with a 7683series auto 2sam pler 1The peaks were identified by reference to a mass spectral library 1R esults T otally 352sam ples were collected from the netw ork collaborating hospitals since 20011Thirty 2four (9166%)cases of various inherited metabolic diseases were diagnosed with an age range of 2days to 14years 1The disease profile was consisted of methylmalonic acidemias (6),α2keto 2glutaric aciduria (5),tyrosinemia type Ⅰ(4),dicarboxylic aciduria (4),multiple carboxylase deficiency (3),phenylketonuria (3),lactic acidemia (3),propionic acidemia (2),ornithine transcarbam oylase deficiency (1),ethylmalonic 2adipic aciduria (1),glutaric aciduria type Ⅱ(1)and 32methylcrotyl C oA carboxylase deficiency (1)1The m ost comm on clinical sym ptoms and signs included mental and developmental retardation ,convulsion ,musculotonic abnormality and jaundice 1R outine laboratory tests often revealed metabolic acidosis ,hypoglycemia and hyperamm onemia ,etc 1Conclusion Urine organic acids analysis by G C 2MS remains to be the m ost important technique for the high 2risk screening and diagnosis of inherited metabolic diseases 1Use of urine filter paper for sample collection and analysis in advanced genetic metabolic centers is a practical approach to extend the diagnostic capacity and improve the management of such diseases in China 1C ollaborative netw ork played a critical role in thesuccess of the program1【K ey w ords】　Metabolism,intorn errors;　Organic acids;　Mass fragmentograpny;　Urinalysis 遗传性代谢病是一类重要的遗传性疾病,虽单一病种的发病率低,但其作为一类疾病的总体发病率甚高,目前已经认识的遗传性代谢病已超过500种[1]。

近年来生物化学、分子遗传学和计算机科学的迅速发展,为遗传性代谢病的诊治和研究提供了良好契机。

随着人类疾病谱的改变,遗传因素在人类疾病发病中的相对作用日趋重要,遗传性代谢病的诊断和治疗在我国亦逐步引起重视[224]。

自1966年T anaka等[5]应用气相色谱2质谱联用技术(gas chromatography2mass spectrometry,G C2MS)诊断首例异戊酸血症以后,检测技术和方法不断改进,迄今仍为遗传性代谢病最重要的诊断手段。

既往我国对此类疾病仅能作出初步诊断[6],或依靠将标本送至国外合作单位确诊[7210]。

我们于2000年在国内率先独立建立了尿有机酸、氨基酸及其他代谢产物的G C2MS分析方法,并成立了全国性遗传代谢性疾病高危筛查诊断协作网络,采用滤纸收集尿液标本,系统开展了遗传性代谢病的高危筛查诊断工作。

自2001年4月开始检测标本,至2002年10月已分析样本352例。

报告如下。

资料和方法一、材料及标本来源1.材料:所有有机酸标准品及饱和烃(C24)均购自美国Sigma公司,选取十五烷酸和22酮己酸为内标,C24为外标。

乙酸乙酯、乙醚和正己烷购自美国Fisher公司,甲基硅烷化衍生剂(BSTFA+1%T MCS)由美国Supelco公司提供。

常规生化试剂为上海化学试剂总厂分析纯产品,高纯氦气和氮气(991999%)为武汉钢铁公司产品。

2.标本来源:(1)同济医院儿科门诊、病房。

(2)遗传代谢性疾病高危筛查诊断协作网络单位。

为深入开展遗传性代谢病的筛查诊断工作,充分发挥各地区医疗单位的技术和资源优势,加强合作,提高对此类疾病的诊断治疗整体水平,在中华医学会儿科学分会内分泌遗传代谢性疾病学组各单位的支持下,来自国内20余家医疗单位的代表于2001年4月在武汉同济医院成立了遗传代谢性疾病高危筛查诊断协作网络并为本研究提供部分标本。