滤纸尿半乳糖醇气相色谱/质谱分析在筛查希特林蛋白缺陷致新生儿肝内胆汁淤积症中的应用

目的观察不同大小及不同放置时间的滤纸洗脱尿半乳糖醇气相色谱/质谱(GC/MS)检测结果与原尿液半乳糖醇GC/MS检测结果之间差异,探讨滤纸尿送检筛查希特林蛋白缺陷所致新生儿肝内胆汁淤积症(NICCD)的可行性。方法选择既往GC/MS分析提示含有半乳糖醇的原尿液10份,每份制成3种大小不同的滤纸吸附,晾干,在室温下放置1～7 d,然后洗脱。取原尿液和洗脱尿100μL,用生化分析仪测定尿肌酐水平。取原尿液和洗脱尿100μL,原尿液先采用尿素酶处理后进行尿液代谢产物GC/MS检测。用质谱仪检索NIST图谱库定性检测到的尿液代谢产物。用正十七烷酸作为内标物质,应用内标法以内生肌酐定量代谢产物。结果同一份含有半乳糖醇的尿液,制成3种大小不同的滤纸尿,放置第1、3、5、7天的大小不同滤纸洗脱尿与其原尿液均经GC/MS检测,并进行比较,发现不同时间/不同大小的滤纸洗脱尿与其原尿液半乳糖醇的出峰时间及水平,均无统计学差异,不同时间/不同大小的滤纸洗脱尿半乳糖醇的出峰时间及水平均无统计学差异。结论在常温下,7 d内当洗涤滤纸的液体与滤纸吸收的尿液量一致时,滤纸尿可代替-80℃储存的原尿。建议使用的滤纸片规格是5 cm×8 cm,可吸收1.0 mL尿液。

Clinical Value of Galactitol Concentration in Urine Filter Paper Analyzed by Gas Chromatography - Mass Spectrometry in Screening Neonatal Intrahepatic Cholestasis Caused by Citrin Deficiency

Objective To observe the difference of galactitol concentration analyzed by gas chromatography-mass spectrometry(GC/MS) on different sizes and different duration times between dried urine filter paper samples and original urine samples,and to find the possibi-lity of transporting the samples and screening in diagnosis of patients with neonatal intrahepatic cholestasis caused by citrin deficiency(NICCD)by using dried filter paper. Methods Ten galactitol positive original urine samples diagnosed by GC/MS were collected,each sample was absorbed with 3 different sizes of filter paper,then dried and placed under the room temperature for 1-7 days,then elevated.One hundred microliter original urine or filter paper elution were collected and the concentration of the urine creatinine was measured by biochemical met-hod.One hundred microliter original urine or filter paper elution were collected and metabolin was measured with GC/MS after urea had been removed by urease.Nature of the metabolin obtained from the urine were determined by searching through the NIST bank with mass spectrometer.The quantity of metabolin was measured by endogenous creatinine by using interior label method with heptadecane acid as a standard. Results Three different sizes of urine filter paper samples were made with the urine sample which contained galactitol,collected from the same patient.After being placed at room temperature for 1,3,5 or 7 days,when tested by GC/MS,there were no significant differences on time to get to the peak concentration and content of galactitol between different sizes or different time points of urine filter paper sample and the original urine samples.There were no significant differences on time to get to the peak concentration and content of galactitol among different sizes or different time points of urine filter paper samples,too. Conclusions At the room temperature,when the volume of elution is equal to the volume of urine absorbed by the filter paper and the duration time is less than 7 days,deep froze(-80 ℃) original urine sample can be replaced by dried urine filter paper sample.The size of the filter paper shall be 5 cm×8 cm,and it can absorb 1.0 mL urine.