液相串联质谱在遗传代谢病高危新生儿检测中运用研究

目的研究液相串联质谱在遗传代谢病高危新生儿检测中的意义。方法留取560例临床疑似遗传代谢病新生儿的干血滤纸片,经含氨基酸、酰基肉碱内标的甲醇萃取,盐酸正丁醇衍生后,进行液相串联质谱分析。结果560例高危新生儿中检测出患儿48例,阳性率8．6％（48／560）,包括脂肪酸代谢病11例（22．9％）;氨基酸代谢病15例（31．3％）;有机酸代谢病22例（45．8％）。结论液相串联质谱可以通过检测血滤纸片中的不同酰基肉碱浓度,一次分析快速检测30多种遗传代谢病,对于遗传代谢病高危新生儿的临床诊断具有重要的价值。     

串联质谱技术在新生儿遗传代谢病筛查中的应用

遗传代谢病是一组种类繁多,临床症状复杂的疾病,常累及神经系统等多器官,一旦发病危害严重。通过新生儿筛查技术早发现早治疗是预防遗传代谢病危害的有效途径。现对串联质谱技术在新生儿遗传代谢病筛查应用中的技术原理、实验流程、面临的挑战和问题作综述。     

儿童扩张性心肌病伴脂肪酸氧化代谢异常9例

目的探讨儿童扩张性心肌病伴脂肪酸氧化代谢异常的临床特征、诊断与治疗。方法回顾性分析2007年1月至2011年6月在首都医科大学附属北京安贞医院诊断为扩张性心肌病伴脂肪酸氧化代谢异常患儿的临床特征、实验室检查、治疗和随访情况。结果 9例扩张性心肌病伴脂肪酸氧化代谢异常患儿进入分析,男5例,女4例。病程0.5-4.5年,起病年龄11个月至18岁。9例均有乏力表现,4例合并肌肉无力,1例四肢近端肌萎缩,2例有惊厥发作,2例伴生长发育落后。9例均有不同程度肝肿大,8例肝功能异常,7例CK、CK-MB和乳酸脱氢酶增高,6例乳酸增高,2例低血糖,3例高血氨。9例超声心动图检查均可见左心室舒张末期内径增高和射血分数下降。肌电图示肌源性损害2例。串联质谱检查示游离肉碱明显增高1例,减少5例,正常3例;酯酰肉碱增高7例,减少1例,正常1例。临床诊断肉毒碱棕榈酰转移酶Ⅰ缺乏症1例,原发性肉碱缺乏症1例,多种酰基辅酶A脱氢酶缺乏4例,长链酰基辅酶A脱氢酶缺乏3例。9例在常规抗心力衰竭治疗基础上,补充左旋肉碱、维生素及低脂肪、预防饥饿等饮食指导。随访0.5-3年,临床症状、生化指标和超声心动图表现逐渐改善。结论原因不明的心脏扩大合并多系统症状和生化指标异常者,应考虑到脂肪酸代谢异常可能,及早进行代谢病筛查,早期诊断和合理治疗是改善预后的关键。     

串联质谱联合二代测序技术在陕西地区遗传代谢病诊断中的应用

目的 探讨串联质谱联合二代测序技术在遗传代谢病(inborn errors of metabolism,IEM)诊断中的应用价值。 方法 采用串联质谱技术对2018年1月至2019年12月西北妇女儿童医院新生儿筛查中心的60 272例新生儿进行遗传代谢病筛查,对其中34例阳性患者采用高通量测序技术进行遗传学分析。 结果 39例患者被确诊为遗传代谢病,包括氨基酸代谢病18例(46.15%),有机酸代谢病15例(38.46%)和脂肪酸氧化障碍类疾病6例(15.38%),本地区IEMs发生率为1∶1545。高苯丙氨酸血症和甲基丙二酸血症是两种最常见的遗传代谢病,占所有确诊病例的66.67%(26/39)。高苯丙氨酸血症的 PAH基因c.728G>A和c.721C>T、甲基丙二酸血症的 MMACHC基因c.567dupT和c.609G>A、 MMUT基因c.323G>A和c．1630_1631delGGinsTA以及希特林蛋白缺乏症的 SLC25A13基因c.852_855del是常见的突变位点。 结论 串联质谱新生儿筛查与二代测序技术的联合应用能够有效地提高遗传代谢病的诊断效率。遗传代谢病在陕西地区并不罕见,初步呈现了本地区遗传代谢病的发病率、疾病谱及遗传特征,为串联质谱技术在陕西地区新生儿筛查中的推广应用及基因检测的临床实践奠定基础。     

串联质谱与二代测序联用在新生儿遗传代谢病诊断中的应用价值

目的 探究串联质谱与二代测序联用在新生儿遗传代谢病诊断中的应用价值.方法 回顾性分析2018年6月至2020年12月山东省妇幼保健院收治的临床表现疑似遗传代谢病高危新生儿32例为研究对象,所有患儿均给予串联质谱与二代测序筛查遗传代谢病.结果 32例临床表现疑似遗传代谢病新生儿,均先行串联质谱检测,后经测序技术检测后诊断...     

4819例遗传代谢病检测结果分析

目的 为了解遗传代谢病的发病率,以便推动遗传代谢病的全面筛查,应用液相色谱-串联质谱(liquid chromatography-tandem mass spectrometry,LC-MS/MS)检测血氨基酸和酰基肉碱,联合气相色谱-串联质谱(gas chromatography-tandem mass spectrometry,GC-MS)技术检测尿液中有机酸,对氨基酸、有机酸代谢病及脂肪酸β氧化障碍进行筛查和诊断。 方法 收集4819例(包括1388例新生儿及3431例疑似遗传代谢病高危儿童)血氨基酸和酰基肉碱检测结果及尿有机酸检测结果,分别利用LC-MS/MS检测了4778例干滤纸片和GC-MS检测了3004例尿标本。 结果 通过遗传代谢病筛查共确诊88例(占所检测样本的1.83%,这88例均行LC-MS/MS和GC-MS检测),其中氨基酸代谢病9种,37例;有机酸代谢病7种,40例;脂肪酸β氧化障碍5种,11例。 结论 联合LC-MS/MS及GC-MS能快速对遗传代谢病进行筛查和诊断。     

两种非衍生化串联质谱检测试剂在新生儿遗传代谢病筛查中的应用比较

目的 比较两种非衍生化串联质谱检测试剂(NeoBase™ 1和NeoBase™ 2)在新生儿遗传代谢病筛查中的应用情况。 方法 选择2020年6月至12月在西北妇女儿童医院进行遗传代谢性疾病筛查的表型正常新生儿滤纸干血片标本,共计7295份。同时应用NeoBase™ 1和NeoBase™ 2试剂检测滤纸干血斑样本,了解正常新生儿11种氨基酸和12种酰基肉碱水平分布,并制定参考区间;采用配对Wilcoxon检验比较11种氨基酸和12种酰基肉碱水平之间差异;同时检测经临床确诊的23例6种遗传代谢病患儿的标本,验证两种试剂对疾病判读的一致性。 结果 正常新生儿的11种氨基酸和12种肉碱水平经K-S单标本正态分布检验,呈偏态分布(均 P<0.001),两种试剂检测的所有指标水平差异均有统计学意义(均 P<0.001);以0.5~99.5百分位数确定两种试剂检测各种氨基酸和酰基肉碱的正常参考范围,两种试剂确定的参考范围有重叠,经临床确诊样本验证,NeoBase™ 2与NeoBase™ 1均检测到相应异常指标的改变,两种试剂均能检测出疾病患儿,检出率均为100%。 结论 NeoBase™ 2与NeoBase™ 1相比,检测氨基酸和酰基肉碱的水平有差异,但均能检测到确诊样本的异常指标改变,对疾病的判断无影响,可用于新生儿遗传代谢性疾病的筛查。     

廊坊地区41062例新生儿遗传代谢病串联质谱筛查结果分析

目的 对廊坊地区41062例新生儿35种遗传代谢病串联质谱筛查情况进行总结分析。方法 对2020年1月至2020年12月廊坊地区出生的新生儿足跟血进行氨基酸代谢异常、有机酸代谢异常、脂肪酸代谢异常等35种遗传代谢病串联质谱检测,总结目前廊坊地区新生儿串联质谱筛查工作现状,比较廊坊地区与国内外其他地区遗传代谢病发病率及种类分布。结果 41062例新生儿中,共筛查出患病儿童18例,总检出率为1/2281,其中氨基酸代谢障碍性疾病9例（50%）,脂肪酸代谢障碍性疾病5例（27.78%）,有机酸代谢障碍性疾病4例（22.22%）。结论 串联质谱检测技术可以高效地对遗传代谢病进行筛查,做到早发现早诊断早治疗,是出生缺陷防控的重要措施。     

干滤纸血片制备因素对遗传代谢病串联质谱分析结果的影响

目的 探究新生儿遗传代谢病筛查实验室分析前环节中,干滤纸血片制备时可能影响串联质谱分析结果的因素。方法 将所收集全血的红细胞压积调整为50%～55%,向其中富集瓜氨酸、甲硫氨酸、苯丙氨酸、游离肉碱、乙酰肉碱、丙酰肉碱工作液,用移液枪吸取后滴落至滤纸上,将干滤纸血片样本按血斑点样血量(10、25、35、50、75、100μL)分6组,测量各组血斑直径。采用LC-MS/MS方法分析25、35、50、75、100μL组样本内主要氨基酸(甲硫氨酸、苯丙氨酸、瓜氨酸)和酰基肉碱(游离肉碱、乙酰肉碱、丙酰肉碱)标志物浓度水平。比较50μL组血片样本不同部位(中央和边缘)的主要标志物浓度变化。结果 直径均大于8mm的血斑样本组主要氨基酸、酰基肉碱浓度比较存在差异。25μL组样本内主要标志物浓度均显著小于50μL组、75μL组、100μL组(P均<0.05);50μL组和75μL组样本内各标志物水平相当,差异未见统计学意义;100μL组样本中主要标志物浓度均显著大于其余各组(P均<0.05)。在干血片边缘部位的主要标志物浓度水平高于中央部位,差异有统计学意义((P<0.05)。结论 在氨基酸和酰基肉碱谱的实验室分析前环节中,干滤纸血片制备时不同血斑点样血量和取样部位会对串联质谱分析结果造成影响,在制备血片时,推荐点样血量为50～75μL,取样部位应选取在血斑边缘部位进行。     

串联质谱与NGS在遗传代谢病诊断性能评估对比及评估其影响因素

目的 比较串联质谱检测与二代测序技术（NGS）诊断疑似遗传代谢病（IMD）的新生儿的性能,并分析影响其诊断的可能因素。方法 纳入2019年8月至2022年6月承德市妇幼保健院临床表现疑似IMD的120例新生儿,均接受参加串联质谱检测和NGS诊断。统计分析不同诊断方法的IMD分类情况,比较两种方法的诊断效能。比较不同胎龄（早产、足月）、出生体质量（≤2500 g、>2500 g）、采血时间（3～7 d、>7 d）新生儿氨基酸浓度。结果 120例疑似IMD新生儿中,经串联质谱诊断判断阳性病例有27例（22.50%）,其中氨基酸代谢病13例、脂肪酸代谢病6例、有机酸代谢病8例。120例疑似IMD新生儿中,经NGS诊断判断阳性病例有25例（20.83%）,其中氨基酸代谢病12例、脂肪酸代谢病7例、有机酸代谢病6例。以NGS测序结果为金标准,串联质谱诊断准确率为93.33%(112/120),敏感度、特异度分别为88.00%(22/25)、94.74%(90/95)。不同胎龄（早产、足月）的丙氨酸（Ala）、甘氨酸（Gly）、甲硫氨酸（Met）、鸟氨酸（Orn）、脯氨酸（Pro）、亮...     

First prenatal diagnosis of the carnitine transporter defect

We report the first attempt at prenatal diagnosis of the carnitine transporter defect in a fetus at high risk of having the disorder. Analysis of cultured CVS after prolonged culture predicted that the fetus was not affected but might be heterozygous for the carnitine transporter defect, but chromosome 15 satellite DNA markers showed no paternal contribution, suggesting that the CVS cells assayed were of predominantly maternal origin. Subsequent assay of cultured amniocytes predicted that the fetus would be affected, and this was confirmed in the newborn period. We conclude that prenatal diagnosis of the carnitine transporter defect is possible, but where results depend on extended culture of CVS, molecular studies should be performed to confirm genetic contributions from both parents. © 1996 Wiley-Liss, Inc.     

D-Amino acids in rat brain measured by liquid chromatography/tandem mass spectrometry

Previous work has established that D-amino acids including D-serine (D-Ser) and D-aspartic acid (D-Asp) fulfill specific biological functions in the brain. In this work, the levels and anatomical distribution of d-amino acids in rat brain were determined by using an advantageous liquid chromatography/tandem mass spectrometric analytical method. The study was focused on D-Ser, D-Asp, and D-glutamic acid (D-Glu) because of the significance of L-Asp, L-Glu, and D-Ser in the nervous system. Prenatal, postnatal pups, and 90-day old rats were studied. Results indicated that D-Asp and D-Ser occurred in rat brain at the microg/g tissue level. However, D-Glu was not detected (< 110 ng/g tissue). Throughout the developmental stages d-Asp content in rat brain decreased rapidly from 9.42% of total Asp in 5-day prenatal rats to an undetectable level (< 150 ng/g tissue) in 90-day old rats. In contrast, D-Ser level increased gradually throughout the developmental stages. D-Ser percentage (D-Ser/(D-Ser + L-Ser)) changed from 4.94% in 5-day prenatal rats to 13.7% in 90-day old rats. Regional levels of D-Ser were found to be significantly higher in cortex, striatum, and hippocampus than in thalamus. D-Ser was not detected in cerebellum (< 172ng/g tissue).     

Evaluation of urinary acylglycines by electrospray tandem mass spectrometry in mitochondrial energy metabolism defects and organic acidurias

We analyzed the urinary acylglycine excretion in 26 patients with mitochondrial energy metabolism disorders and in 55 patients with organic acidurias by electrospray tandem mass spectrometry (ESI-MS/MS), monitoring precursor ions of m/z 90. Urinary concentrations of the different acylglycines were quantified using deuterated internal standards. Normal values for the most important acylglycines were established. In MCAD and MAD (neonatal form) deficiencies, typical excretion patterns of urinary acylglycines were found in all the samples. In isovaleric aciduria, propionic aciduria, and 3-methylcrotonylglycinuria typical glycine conjugates were always found. Methylmalonic aciduria (mutase deficiency), multiple carboxylase deficiency, and 3-hydroxy-3-methylglutaric aciduria revealed pathological acylglycine profiles, even if not specific for the disease. In all these diseases acylglycine excretion seems to be less influenced by the clinical status than organic acid excretion. This method is a useful diagnostic tool for these metabolic disorders, complementary to organic acids and acylcarnitine profiles.     

Contribution of FAT/CD36 to the regulation of skeletal muscle fatty acid oxidation: an overview

Long chain fatty acids (LCFAs) are an important substrate for ATP production within the skeletal muscle. The process of LCFA delivery from adipose tissue to muscle mitochondria involves many regulatory steps. Recently, it has been recognized that LCFA oxidation is not only dependent on LCFA delivery to the muscle, but also on regulatory steps within the muscle. Increasing selected fatty acid binding proteins/transporters on the plasma membrane facilitates a very rapid LCFA increase into the muscle, independent of any changes in LCFA delivery to the muscle. Such a mechanism of LCFA transporter translocation is activated by muscle contraction. Intramuscular triacylglycerols may also be hydrolysed to provide fatty acids for mitochondrial oxidation, particularly during exercise, when hormone-sensitive lipase and other enzymes are activated. Mitochondrial LCFA entry is also highly regulated. This however does not involve only the malonyl CoA carnitine palmitoyltransferase-I (CPTI) axis. Exercise-induced fatty acid entry into mitochondria is also regulated by at least one of the proteins (FAT/CD36) that also regulates plasma membrane fatty acid transport. Among individuals, differences in mitochondrial fatty acid oxidation appear to be correlated with the content of mitochondrial CPTI and FAT/CD36. This paper provides a brief overview of mechanisms that regulate LCFA uptake and oxidation in skeletal muscle during exercise and in obesity. We focus largely on our own work on FAT/CD36, which contributes to regulating, in a coordinated fashion, LCFA uptake across the plasma membrane and the mitochondrial membrane. Very little is known about the roles of FATP1-6 on fatty acid transport in skeletal muscle.     

Nanostructure-initiator mass spectrometry (NIMS) imaging of brain cholesterol metabolites in Smith-Lemli-Opitz syndrome

Cholesterol is an essential component of cellular membranes that is required for normal lipid organization and cell signaling. While the mechanisms associated with maintaining cholesterol homeostasis in the plasma and peripheral tissues have been well studied, the role and regulation of cholesterol biosynthesis in normal brain function and development have proven much more challenging to investigate. Smith–Lemli–Opitz syndrome (SLOS) is a disorder of cholesterol synthesis characterized by mutations of 7-dehydrocholesterol reductase (DHCR7) that impair the reduction of 7-dehydrocholesterol (7DHC) to cholesterol and lead to neurocognitive deficits, including cerebellar hypoplasia and austism behaviors. Here we have used a novel mass spectrometry-based imaging technique called cation-enhanced nanostructure-initiator mass spectrometry (NIMS) for the in situ detection of intact cholesterol molecules from biological tissues. We provide the first images of brain sterol localization in a mouse model for SLOS (Dhcr7^−/−). In SLOS mice, there is a striking localization of both 7DHC and residual cholesterol in the abnormally developing cerebellum and brainstem. In contrast, the distribution of cholesterol in 1-day old healthy pups was diffuse throughout the cerebrum and comparable to that of adult mice. This study represents the first application of NIMS to localize perturbations in metabolism within pathological tissues and demonstrates that abnormal cholesterol biosynthesis may be particularly important for the development of these brain regions.