【作者】 程静；

【导师】 王华东；

【作者基本信息】 暨南大学 ， 病理生理学， 2007， 硕士

【摘要】 目的：拟对NICCD(希特林蛋白缺陷所致的新生儿肝内胆汁淤积症)患儿进行尿半乳糖醇GC／MS(气相色谱／质谱)分析，从而提出筛查NICCD的生化指标，并探讨NICCD基因型与尿半乳糖醇水平之间的关系，进一步观察不同大小及不同放置时间的滤纸洗脱尿半乳糖醇GC／MS检测结果与原尿液半乳糖醇GC／MS检测结果之间的差异，探讨滤纸尿送检筛查NICCD的可行性。方法：1．选择经外周血SLC25A13基因突变分析已明确基因型诊断的15例NICCD患儿，收集他们治疗前后的尿液标本，并收集10例病毒性肝炎急性期患儿，60例正常健康儿童的尿液标本作对照。2．原尿液采用尿素酶前处理后进行GC／MS检测。3．用质谱仪检索NIST图谱库定性检测到的尿液代谢产物。4．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。5．选择既往GC／MS分析示含有半乳糖醇的原尿液10例，每例制成三种大小不同的滤纸吸附，晾干，在室温下放置1～7天，然后洗脱。6．取原尿液和洗脱尿100uL用生化分析仪测定尿肌酐含量。7．取原尿液和洗脱尿100uL同方法2—4处理。8．数据呈偏峰分布，以中位数(M)和95％可信区间(95％CI)表示，采用非参数Kruskal-Wallis test检验，如有统计学意义则采用Mann-Whitney U进一步行组间分析，P＜0.05时认为差异有统计学意义。结果：1．NICCD患儿治疗前尿半乳糖醇水平明显高于正常组儿童和肝炎组患儿；2．NICCD患儿Ⅰ类纯合组、Ⅰ类杂合组治疗前尿半乳糖醇水平无显著性差异；3．Ⅰ类突变纯合组和杂合组治疗后尿半乳糖醇水平均明显下降，与治疗前比较有显著性差异；4．同一例尿液，放置第1天、第3天、第5天和第7天的三种大小滤纸洗脱尿半乳糖醇的含量与原尿半乳糖醇的含量比较，均无显著性差异。结论：1、尿液GC／MS分析发现半乳糖醇水平明显升高时提示患儿可能有NICCD，尿半乳糖醇可以作为筛查NICCD的生化指标。2、尿液GC／MS分析检测半乳糖醇对NICCD的基因分型无明显提示作用。3、在常温下，7天之内当洗涤滤纸的液体与滤纸吸收的尿液量一致时，滤纸尿可以代替-80℃储存的原尿。我们建议用的滤纸片规格是：5cm×8cm，可以吸收1.0mL尿液。更多还原

【Abstract】 Objective:Galactitol concentration in the urine sample of patient with NICCD (neonatal intrahepaticcholestas is caused by citrin deficiency, NICCD ) was analyzed by GC/ MS (Gaschromatography-Mass spectrometry) to establish a biochemical criteria to screen the patientswith NICCD among the population, and the relationship between the genotype of NICCD andgalactitol concentration in the urine was studied. Furthermore, the difference of galactitolconcentration analyzed by GC/MS from different size and different duration time of dried urinefilter paper sample and original urine sample were observed to study the possibility oftransporting the sample by dried urine filter paper in order to screen the patients with NICCD.Methods:1. The urine sample, from 15 patients, whose genotype of NICCD were identified bySLC25A13 genetic mutation analysis of peripheral blood, both pre-therapy and post-therapy, 10patients with virus hepatitis and 60 healthy children, were collected.2. Original urine samples were analyzed by gas chromatography-mass spectrometryafter urea had been removed by urease.3. Metabolites in urine were identified by mass spectrometry and NIST library.4. Heptadecanoic acid was used as internal standard and metabolites concentration inurine were calculated up on creatinine concentration.5. Ten galatitol positive original urine samples analyzed by GC/MS were collected.6. Each sample was absorbed by 3 different size of filter paper, then dried and placeunder the room temperature for 1～7 days, then eluated.7. 100μL sample of original urine and elution was collected to measure theconcentration of the urin creatinine by biochemical method.8.100μL of original urine sample and elution sample were collected and measuredas method 2-4.9. Non-normal distributed data presented as media(M) and 95%confidenceinterval(95%CI). Statistical evaluation for nonparametric data was carried out using theKruskal-Wallis test, if there was statistical significance , Mann-Whitney U test was used toevaluate the difference between two groups. The level of significance was P＜0.05.Results:1.Galactitol concentration in the urine sample before therapy in patient with NICCDhas significant difference with patients with virus hepatitis and healthy people; 2. There was no significant differences in galactitol concentration in the urin samplebefore therapy between patient with type I homozygote NICCD and type I heterozygoteNICCD.3. There was a significant difference in galactitol concentration in the urin samplebetween pre-therapy and post-therapy in patients with type I homozygote NICCD or type Iheterozygote NICCD.4. Urine filter paper sample collected from same patient, compared with original urine,the galactitol concentration in the urine has no significant differences after being placed in roomtemperature for 1, 3, 5or7 days.Conclusion:1. An marked increase in galactitol concentration analyzed by GC-MS in the urine sampleusually means the possibility of NICCD.2. Galactitol concentration analyzed by GC-MS in the urine sample has no use inidentifying the genotype of NICCD.3. At the room temperature, when the volume of elution is equal to the volume of urineabsorbed by the filter paper and the duration time is less than 7 days, deep froze original urinesample can be replaced by urine filter paper sample. The size of the filter paper should be: 5cm×8cm, and it can absorb 1.0 mL urine.更多还原