半乳糖血症患儿尿液气相色谱-质谱结果分析

目的 应用气相色谱-质谱（GC-MS）技术检测10例半乳糖血症患儿治疗前后尿液，以建立半乳糖血症的化学诊断方法。方法 留取10例半乳糖血症患儿治疗前后尿液和60例同年龄段正常儿童尿液，尿液经尿素酶处理后用GC-MS检测尿代谢产物。结果 半乳糖血症患儿尿中检测出至少34种代谢物，其中半乳糖、半乳糖醇、半乳糖酸、酪氨酸、4-羟基苯乙酸、4-羟基苯乳酸水平均较正常对照组明显升高（P均〈0．05），二氢尿嘧啶水平较正常对照组显著降低（P〈0．05），治疗后除4-羟基苯乳酸外，其他均恢复至正常范围。结论 GC-MS检测尿中半乳糖、半乳糖醇、半乳糖酸水平可诊断半乳糖血症。     

尿素酶预处理-气相色谱-质谱法选择性筛查遗传代谢病高危患儿327例初步研究

目的　通过对遗传代谢病高危患儿尿液成分进行生化分析 ,筛查遗传代谢病 ,为临床诊断和治疗提供实验依据。方法　收集遗传代谢病高危患儿尿液标本 ,经去尿素、加内标、除蛋白、真空干燥、三甲基硅烷基衍生等处理后 ,应用气相色谱 -质谱联用仪分析尿液中有机酸、氨基酸、糖类、多醇、嘌呤、嘧啶等成分。这一流程在国际上被称为尿素酶预处理 气相色谱 质谱法。结果　对来自中国大陆 6省、区和直辖市的 3 2 7例遗传代谢病高危患儿的尿液标本进行检测 ,共筛查出遗传代谢病 16种 2 7例 ,阳性率为 8.2 6% ,其中高苯丙氨酸血症、甘油尿症和Leigh综合征各 3例 ,丙酸血症、甲基丙二酸尿症、vonGierke病、果糖 1,6 二磷酸酶缺陷病、果糖尿症各 2例 ,多种羧化酶缺陷病、戊二酸血症Ⅰ型、枫糖尿病、高甘氨酸血症、3 氨基异丁酸尿症、半乳糖血症、瓜氨酸血症Ⅱ型及Fanconi综合征各 1例。经临床干预虽然仍有部分患儿预后不良 ,但多种羧化酶缺陷病、甲基丙二酸尿症、半乳糖血症等患儿获得较好的治疗效果。其余患儿的病情有待追踪观察。结论　应用尿素酶预处理 气相色谱 质谱法分析尿液成分 ,是筛查某些遗传代谢病的有效方法 ,检测结果可为患儿的诊断和治疗提供有效指导。     

先天性遗传代谢病的早期诊断

目的提高儿科医生对新生儿期遗传代谢病的认识,做到早期诊断、早期治疗.方法自2003年9月至2004年9月,根据临床表现确定18名遗传代谢病高危患儿,用"滤纸片代"将采集的尿标本外寄进行气相色谱-质谱(GC/MS)分析,筛查遗传代谢病.结果 18例高危儿中确诊为遗传代谢病5例,分别为戊二酸尿症Ⅱ型1例(46h,男),鸟氨酸氨甲酰转移酶缺陷1例(66h,男),枫糖尿病1例(8 d,男),甲基丙二酸血症1例(13 d,男),丙酸血症1例(21 d,女),并对其临床特点进行归纳总结.结论掌握新生儿遗传代谢病临床特点,对高危儿早期进行尿GC/MS分析,可以早期诊断遗传代谢病,有利于优生优育.     

气相色谱-质谱法对高危婴幼儿遗传性代谢疾病筛查的研究

目的：研究气相色谱－质谱法对高危婴幼儿遗传性代谢疾病筛查的意义的价值,并对阳性患儿的临床特点进行分析。方法：对705名原因不明的智力－运动发育迟缓、倒退及疑有遗传代谢病的婴幼儿,采用气相色谱－质谱（GC－MS）方法进行尿液化学分析。结果：705例中尿化学分析异常者65例（9．2％）,均为遗传性代谢疾病患儿,其中半乳糖血症11例,苯丙酮尿症、甲基丙二酸尿症各10例,范可尼综合征3例、戍二酸、赖氨酸尿症、疑似同型丝氨酸尿症各2例,果糖1．6二磷酸酶缺乏症、焦谷氨酸尿症、长链脂肪酸尿症及神经母细胞瘤、鸟氨酸尿症、多羧酸酶缺乏症各1例,单纯性糖尿2例,糖尿病2例,乳酸及酮尿症15例。对筛查出的数种疾病患儿进行合理的治疗均取得明显效果。结论：（1）对不明原因的智力－运动发育及倒退的婴幼儿应进行遗传性代谢疾病的筛查。（2）GC－MS检测技术方法准确,敏感性高,特异性强,是筛查遗传性代谢疾病的有效手段。（3）婴幼儿遗传性代谢疾病的筛查应尽早进行,扩大新生儿遗传性代谢疾病筛查的覆盖面,增加筛查病种是非常重要的。     

半乳糖缺陷IgA1在儿童紫癜性肾炎早期诊断中的意义

目的 探讨半乳糖缺陷IgA1（Gd-IgA1）在儿童紫癜性肾炎（HSPN）早期诊断中的价值。方法 以2018年1~4月确诊为HSPN的住院患儿67例、过敏性紫癜（HSP）住院患儿58例为研究对象,另选取20例行健康体检儿童作为健康对照组。采用ELISA法检测血清、尿液Gd-IgA1水平。以受试者工作特征曲线（ROC）分析血清Gd-IgA1及尿液Gd-IgA1/尿肌酐比值对HSPN的诊断价值。结果 HSP及HSPN患儿血清Gd-IgA1、尿液Gd-IgA1/尿肌酐比值均高于健康对照组（P < 0.01）,且以HSPN患儿升高更明显（P < 0.01）。血清Gd-IgA1水平≥ 1 485.57 U/mL和/或尿液Gd-IgA1/尿肌酐比值≥ 105.74时,对诊断HSPN有较好的意义。49例HSP患儿随访6个月,随访中HSPN发病率为47%（23/49）;其中,血清Gd-IgA1 ≥ 1 485.57 U/mL患儿HSPN发病率为100%,尿液Gd-IgA1/尿肌酐比值≥ 105.74患儿HSPN发病率为73%。结论 血清及尿液GdIgA1对HSPN的早期诊断有较好的临床价值。     

新生儿有机酸血症4例

目的探讨新生儿期有机酸血症的临床特点和诊疗措施。方法应用串联质谱法(MS/MS)对4例患儿血/尿样本进行有机酸分析和筛查诊断。其中3例确诊为有机酸血症,1例为高度疑似病例。结果经常规纠正酸中毒、稳定内环境治疗无明显改善,4例均在新生儿期死亡。结论有机酸血症是一种临床表现多样、病情变化较快的遗传代谢性疾病。气相色谱法(GC/MS)尿有机酸分析是有机酸血症诊断的可靠方法,而MS/MS则对新生儿期代谢性疾病的筛查具有重要意义。     

轮状病毒肠炎患儿尿半乳糖测定的意义

目的 了解轮状病毒肠炎患儿乳糖不耐受的发生率及乳糖不耐受者的病情变化.方法 收集4～20个月大便轮状病毒检测均阳性的腹泻患儿30例.按要求留取尿标本检测,采用半乳糖氧化酶法测定尿半乳糖,同时记录腹泻患儿的临床症状.治疗7 d追踪腹泻症状变化情况.结果 轮状病毒肠炎患儿约56.67%(17/30例)尿半乳糖试验阳性.尿半乳糖试验阳性患儿腹泻病程较尿半乳糖试验阴性患儿略延长,但二组比较无统计学差异.结论 尿半乳糖测定可检测轮状病毒肠炎患儿乳糖酶缺乏情况,为指导临床治疗提供依据.     

尿素酶预处理-气相色谱-质谱技术筛查遗传性代谢病高危儿

目的 通过尿素酶预处理-气相色谱-质谱法(UP-GC-MS)检测1330例高危儿遗传性代谢病(IMD)及代谢紊乱的发病率,为临床诊断和治疗提供依据.方法 留取出生1d～3岁高危儿尿液样本,尿液标本经尿素酶去尿素、加内标、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/三甲基氯硅烷衍生后进样,应用气相色谱-质谱联用仪分析其尿液中有机酸、氨基酸、糖类、多醇、嘌呤和嘧啶等成分.结果 1330例高危儿中,共890例(66.9％)存在代谢异常.其中确诊IMD 21例(1.6％),包括甲基丙二酸尿症8例,高苯丙氨酸血症3例,尿素循环障碍、半乳糖血症、枫糖尿症、异戊酸血症和丙酸血症各2例;疑似IMD 49例(3.7％),包括酪氨酸血症23例、脂肪酸代谢障碍12例、尿素循环异常8例和Citrin缺陷症6例,其中4例经过串联质谱检测和基因分析得到确诊;确诊非遗传因素所致代谢病40例(3.0％),包括乳酸血症28例及甘油尿症12例;此外,还有780例(58.6％)高危儿存在代谢紊乱,表现为尿半乳糖、4羟基苯乳酸、N-乙酰酪氨酸、乳酸、乳糖、琥珀酸、酮性双羧酸水平增高以及丝氨酸/苏氨酸比例异常.结论 UP-GC-MS是诊断小儿IMD和代谢紊乱的有效方法,必要时应联合应用串联质谱检测及基因分析进行诊断.     

问：串联质谱仪和气质联用仪应用于遗传代谢病筛查诊断时各有哪些特点？

答：串联质谱仪应用于遗传代谢病筛查诊断,检测速度快,2min就可以检测一个标本,主要检测有机酸、氨基酸和脂肪酸代谢障碍等疾病,一次检测即可实现30多种疾病的同时诊断,成本相对较低,是大范围筛查的首选工具。根据串联质谱筛查结果可进行进一步的检查,包括GC／MS尿筛查、酶学检查和基因学检查。     

甲基丙二酸尿症cblB型1例及其MMAB基因新突变

cblB缺陷是甲基丙二酸尿症中的罕见类型,该文首次报道1例中国cblB型甲基丙二酸尿症患儿,就其临床经过、血液酯酰肉碱谱、尿液有机酸分析、基因缺陷进行研究.该患儿以代谢性脑病形式起病,液相串联质谱分析显示患儿血液丙酰肉碱显著增高(22.43 μmol/L,参考值 1.0~5.0 μmol/L),丙酰肉碱/乙酰肉碱比值轻度增高(0.51,参考值0.03~0.50),尿液甲基丙二酸(195.41 mmol/mol肌酐,正常值0.2~3.6 mmol/mol肌酐)及其代谢产物浓度增高,血清总同型半胱氨酸浓度正常,符合单纯型甲基丙二酸血症.患儿MUT基因分析未见突变,MMAB基因存在c.562G>A(p.V188M)和c.577G>A(p.E193K)杂合突变,确诊为cblB型甲基丙二酸尿症.其中,c.562G>A为新突变.经羟钴铵肌肉注射、口服左卡尼汀、低蛋白饮食及特殊配方奶粉治疗后,患儿病情逐渐好转.随访至患儿3岁11个月,智力运动明显进步.cblB缺陷患者缺乏特异性症状,临床表型为单纯型甲基丙二酸尿症,可通过代谢筛查及MMAB基因分析获得确诊.     

Simplified screening for organic acidemia using GC/MS and dried urine filter paper: a study on neonatal mass screening

A simplified method for organic acidemia screening using GC/MS, the urease/direct method, is now available. To establish a practical screening system for organic acidemias, we studied the usefulness of dried urine filter paper (filter paper urine) and the application of a personal computer-based system of automated metabolic profiling and interpretation (automated system) that we earlier developed. In a comparison of filter paper urine with liquid urine, creatinine levels ranging from 4.6 to 122.5 mg/dl, showed an excellent correlation (r=0.9554), when the volumes of blotted urine and distilled water soaked were the same. Recovery of all 17 compounds except for citrate was similar between liquid and filter paper urine. CV values of 22 compounds tested ranged from 5.5 to 22.4% in the liquid urine, and from 7.7 to 29.8% in the filter paper urine. The CV values in stable isotope dilution analysis were much smaller in all nine compounds tested. As to the stability of compounds, the percentage changes to values at day 0 were within about +/-25% on day 28. We compiled GC/MS data, including methylene unit values, quantifying and confirming ions of 163 different organic acids and others, to use the automated system. We analyzed specimens from 55 patients with 17 different metabolic disorders. In 54 of the 55 specimens, the correct diagnosis was successfully indicated. Neonatal mass screening for organic acidemias warrants ongoing attention using this simplified method and filter paper urine.     

Stability of urinary HVA and VMA on filter paper

The study examined the stability of HVA and VMA in 1-ml aliquots of a single urine sample stored on filter paper at different temperatures for 2 years. The results showed that HVA and VMA were stable in dried filter paper when stored at 4 °C or lower temperature. Storage at room temperature resulted in degradation of the sample.     

Screening newborns for multiple organic acidurias in dried filter paper urine samples: method development

Screening urine for inherited and acquired organic acidurias in newborns has the potential of preventing severe disease, mental retardation, and death. A method for screening dried urine filter paper samples for acidic markers of at least 20 different metabolic conditions has been developed. These conditions include, among others, maple syrup urine disease; methylmalonic, propionic, isovaleric, glutaric, and hydroxymethylglutaric acidurias; methylcrotonylglycinuria; medium-chain acyl-CoA dehydrogenase deficiency; inherited vitamin responsive disorders B12, biotin, B2), and acquired deficiencies of these vitamins. The preparation of the urine extract is identical to the method we use to screen infants for neuroblastoma. Screening is based on a highly sensitive and specific determination of eight organic acid markers by an automated computerized gas chromatography mass spectrometry system using selected ion monitoring. The markers used for screening are methylmalonic acid, 2-hydroxyisocaproic acid, glutaric acid, propionylglycine, isovalerylglycine, 3-methylcrotonylglycine, hexanoylglycine, and 3-phenylpropionylglycine. The extraction efficiencies of these acids from dried filter paper were similar to extraction from water, ranging from about 40% to 80%, except for propionylglycine which showed a low extraction efficiency of 11-13%. The stability of these acids on filter paper exposed to room air and temperature over a period of 15 d was adequate for the use of this collection method for organic aciduria screening. Normal levels, adjusted to urinary creatinine, were established for these acids in 519 urine filter paper samples obtained from 3-wk-old newborns. This screening method was tested on samples obtained from 12 patients with known organic acidurias including stored urine filter paper collected at 3-wk of age from two infants later found to have organic acidurias.     

先天性遗传代谢病的早期诊断

目的提高儿科医生对新生儿期遗传代谢病的认识，做到早期诊断、早期治疗。方法自2003年9月至2004年9月，根据临床表现确定18名遗传代谢病高危患儿，用“滤纸片代”将采集的尿标本外寄进行气相色谱．质谱(GC／MS)分析，筛查遗传代谢病。结果18例高危儿中确诊为遗传代谢病5例，分别为戊二酸尿症Ⅱ型1例(46h，男)，鸟氨酸氨甲酰转移酶缺陷1例(66h，男)，枫糖尿病1例(8d，男)，甲基丙二酸血症1例(13d，男)，丙酸血症1例(21d，女)，并对其临床特点进行归纳总结。结论掌握新生儿遗传代谢病临床特点，对高危儿早期进行尿GC／MS分析，可以早期诊断遗传代谢病，有利于优生优育。     

Diagnosis of the carbohydrate-deficient glycoprotein syndrome by analysis of transferrin in filter paper blood spots

Carbohydrate-deficient glycoprotein syndrome is a recently identified recessively inherited, multisystemic disease with severe nervous system involvement. It is characterized biochemically by carbohydrate-deficient serum glycoproteins, and can be diagnosed by analysis of abnormal isoforms of serum transferrin. Using stored, neonatally collected filter paper blood spots from such patients, it was shown that neonatal diagnosis was possible by immune-isoelectric focusing of transferrin eluted from up to 14-year-old samples. Freshly collected blood on filter paper was readily analyzed quantitatively for carbohydrate-deficient isotransferrins by a rapid microchromatographic assay, revealing highly elevated values in all patients. The presently described methods thus provide a means for early diagnosis of the carbohydrate-deficient glycoprotein syndrome in microliter volumes of capillary blood. Sampling on filter paper offers an important simplification in sample collection, storage and transport, and may make population studies possible.     

Protein detection in dried blood by surface-enhanced laser desorption/ionization-time of flight mass spectrometry (SELDI-TOF MS)

BACKGROUND: The rapid and reliable identification of biomarkers in the smallest possible amount of blood remains a challenge in biomarker epidemiological research involving preterm newborns. OBJECTIVE: We wanted to explore whether the proteomics approach of 'surface-enhanced laser desorption/ionization-time of flight mass spectrometry' (SELDI-TOF MS) is possible and feasible in whole cord blood previously dried on filter paper. METHODS: Umbilical cord blood from 7 healthy newborns was frozen as serum, whole blood (with or without additives), or dried on filter paper (with or without additives). We used the SELDI-TOF MS technique for protein detection on the ProteinChip arrays: weak cationic exchange array (CM10), hydrophobic array (H50), and strong anion exchange array (Q10). Profiles were compared in terms of peak intensity and number of resolved peaks. Results: Dried neonatal blood, eluted from filter paper, revealed profiles similar to the profiles derived from serum at a protein range of 3-10 kDa. Among additives, heparin led to highest peak intensities for both blood and dried blood. Spectra from heparinized whole blood and heparinized dried blood from the umbilical cord of 8 different healthy newborns on three different types of ProteinChip arrays were very similar. Conclusion: We conclude that it is possible and feasible to use SELDI-TOF MS for recovery and detection of whole proteins from dried blood collected on filter paper. The method is easy to perform in large groups of newborns, minimizing the amount of blood needed for biomarker studies. The validity and reproducibility of this method needs to be studied in detail.     

Determination of EDTA in dried blood samples by tandem mass spectrometry avoids serious errors in newborn screening

Newborn screening programs use whole blood dried on filter paper as the standard specimen. Metabolites are reasonably stable and can easily be sent to screening laboratories by regular mail. The recommended sample collection procedure is to spot native blood without anticoagulants onto the filter paper, because anticoagulants can interfere with the different laboratory methods. However, visual examination of the blood spots cannot always detect contamination. In this study, whole blood was drawn by venous puncture from a healthy volunteer, spiked with the corresponding metabolites and EDTA, and spotted onto filter paper. TSH and 17α-hydroxyprogesterone were determined by time resolved fluoroimmunoassays with the AutoDelfia system. Total galactose, biotinidase activity, and galactose-1-phosphate uridyltransferase activity were measured photometrically or fluorometrically. Succinyl acetone was estimated indirectly through the inhibition of porphobilinogen synthase activity (PBGS assay). EDTA, amino acids, and acylcarnitines were converted to the corresponding butyl esters, after extraction with methanol, and analysed by LC-MS/MS. EDTA contamination gives falsely elevated 17-OHP values and falsely reduced TSH and PBGS values. The inclusion of an EDTA determination in routine screening revealed that at least 0.06% of newborn screening samples were contaminated with EDTA. In conclusion, non-conformity during the pre-analytical phase is a source of false positive and false negative screening results. Determination of EDTA from NBS blood spots can reliably identify these samples and prevent screening errors.     

Metabolic disease in 10 patients with sudden unexpected death in infancy or acute life‐threatening events

In order to determine the associations between sudden unexpected death in infancy (SUDI) or acute life‐threatening events (ALTE) and inborn errors of metabolism, particularly organic acidemia and fatty acid oxidation disorders, we evaluated clinical features in patients with SUDI or ALTE. The subjects were infants between the ages of 7 days and 3 years who developed SUDI or ALTE between January 2004 and December 2013. They were then diagnosed as having inborn errors of metabolism on gas chromatography–mass spectrometry (GC/MS) and/or tandem mass spectrometry (MS/MS). The age distribution, onset forms, and clinical findings were evaluated during the acute phase. Inborn errors of metabolism were detected in three of 196 patients with SUDI, and in seven of 167 patients with ALTE. Of these 10 patients, nine had a history of poor feeding and somnolence during the neonatal period, and symptoms of infection such as cough, fever or vomiting during infancy. Routine laboratory tests during an acute phase indicated hyperammonemia, liver dysfunction, increased blood creatine kinase, acidosis, positive ketone bodies in urine or blood, or hypoglycemia. When SUDI or ALTE are encountered in the emergency unit, it is essential that a detailed medical history is taken, particularly with regard to the neonatal period, and that specific abnormalities are investigated on routine laboratory tests. Moreover, samples such as urine, serum, and filter paper blood specimens should be collected for GC/MS and/or MS/MS of organic acids and acylcarnitines, to identify inborn metabolic disorders.     

Glutamic acid decarboxylase and IA-2 autoantibodies in type 1 diabetes: comparing sample substrates for autoantibody determinations

Large-scale programs designed to assess risk for type 1 diabetes through serologic assessment of autoantibodies to recombinant beta-cell autoantigens are hampered by several limitations, including the methods for sample collection and assay performance, as well as the volume required for autoantibody determinations. The present study was designed to develop a low sample-volume, primary screening method for autoantibody detection of high specificity and sensitivity, and to determine the feasibility of dried blood spots collected on filter paper in serving as vehicles for such determinations. Autoantibodies to glutamic acid decarboxylase (GAD) and ICA512bdc (IA-2), both individually and in combination, were determined in persons with type 1 diabetes, healthy controls, or individuals with other autoimmune disorders. Autoantibody results for serum, plasma, and dried blood spots were compared. GAD, IA-2, and combined GAD/IA-2 autoantibodies were concordant in their measurement from minimal volumes of serum, plasma, and whole blood extracted from dried filter paper. The autoantibody levels from the dried blood spots were, however, lower than corresponding serum samples, and, as currently designed, failed to detect low-titer autoantibodies. Despite this limitation, screening for diabetes risk can be performed using small volumes of whole blood, serum, or plasma collected onto filter paper. These methodological improvements should simplify matters, reduce costs, and increase the efficacy of screening programs for type 1 diabetes. Further development of better substrates/methods for blood-specimen collection seems necessary to exploit the full potential of this and other autoantibody measurement strategies for screening large populations.     

串联质谱技术在脑发育落后病因诊断中的意义

目的探讨串联质谱技术（MS／MS）在脑发育落后病因诊断及疗效判断中的意义。方法应用串联质谱仪,对158例脑发育落后患儿进行血氨基酸谱和酰基肉碱谱定量检测,对检出的11例代谢性疾病患儿MS／MS结果、尿气相色谱／质谱检测（GC／MS）结果、临床表现及治疗后变化进行综合分析。结果158例中,11例（7．0％）患儿确诊为遗传代谢性疾病,其中甲基丙二酸血症5例,丙酸血症2例,鸟氨酸氨甲酰转移酶缺乏症1例,枫糖尿病1例,苯丙酮尿症1例,生物素酶缺乏症1例。临床表现为智能及运动发育落后或倒退（11例）、惊厥（5例）、昏迷（4例）、呕吐（4例）、营养不良（4例）、嗜睡（3例）、反复感染（3例）、肌张力降低（2例）等。实验室检查显示代谢性酸中毒、血氨及血乳酸增高、贫血等。MRI表现为脑萎缩、双侧脑白质T2w高信号或伴T1w低信号、多发性脑软化或囊样变等。起病早、伴严重酸中毒及昏迷的甲基丙二酸血症预后较差。患儿经维生素B12左旋肉碱、特殊奶方、低蛋白饮食及生物素等治疗后,好转8例,死亡3例。结论串联质谱技术有助于脑发育落后的病因诊断及疗效判断。早期诊断及合理治疗可避免脑组织进一步损害,并改善预后。