液相色谱串联质谱法检测法布雷病样本的质量控制研究（英文）

法布雷病(FD)是一种罕见的X连锁隐性遗传的溶酶体贮积病.用于检测FD的常用生物学标志物之一是Lyso-GB3,它是一种脱乙酰基的神经酰胺三己糖苷,通常通过高灵敏和特异性的液相色谱-串联质谱法(LC-MS/MS)进行分析.由于LC-MS/MS技术的复杂性,中国大多数医院仍无法自行建立LC-MS/MS对FD进行高效可靠的诊断.患者的样本需要在当地医院采集,然后送到中心实验室进行LC-MS/MS分析.样本经过漫长的运输和储存过程,可能会影响样本质量,从而影响诊断的准确性.我们首先建立并验证了一种新的LC-MS/MS方法以保证FD样本质量,研究了分析前变量对Lyso-GB3检测的影响.这些分析前变量包括血浆的储存温度和时间、血浆的冻融循环、血浆的溶血程度、全血的储存温度和时间,以及离心温度.最后,我们分析了86个FD样本和100个正常人样本,并对Lyso-GB3浓度和与FD疾病分型的关系进行了相关性研究.结果显示,当血浆在20℃下储存时,Lyso-GB3浓度在4 d后开始下降.此外,溶血会显著影响FD样品的质量,重度溶血导致Lyso-GB3浓度下降了57.8%,其他分析前变量对FD样品质量影响不大.本方法在使用分析前标准化操作流程下,筛查FD的灵敏度和特异度为100%,确保了检测的正确性;当Lyso-GB3浓度的cut-off值为55.15μg/L,鉴别经典型、非经典型患者的灵敏度和特异度为71%和100%,ROC曲线下面积为0.83.我们的研究结果有助于医学实验室操作程序的标准化,以保证FD样本的质量,并为临床判定中国法布雷病的分型提供依据.     

LC-MS/MS法测定大鼠血浆中的野黄芩苷含量

目的:建立LC-MS/MS的分析方法测定大鼠血浆中的野黄芩苷,研究灯盏生脉胶囊中野黄芩苷在大鼠体内的药动学行为。方法:以噻氯匹定为内标,血浆样品经1%甲酸乙腈沉淀蛋白处理后,用LC-MS/MS法测定血浆中的野黄芩苷浓度。结果:野黄芩苷线性范围为1.31～670.00 ng·mL-1(γ0.999),最低定量浓度为1.31 ng·mL-1,回收率、日内、日间考察均符合生物样品分析要求。实验结果显示,野黄芩苷在大鼠体内出现多峰现象。结论:建立的LC-MS/MS定量分析方法灵敏、准确,可用于大鼠血浆中野黄芩苷的测定及其药代动力学研究。     

小型猪血浆中二丙酸倍他米松及其代谢物倍他米松的LC-MS/MS 同时测定方法的建立及在药动学研究中的应用

目的：建立同时测定小型猪血浆中二丙酸倍他米松及其代谢物倍他米松的LC-MS/MS 法,并研究小型猪皮肤外用二丙酸倍他米松乳膏后,二丙酸倍他米松及倍他米松的药动学特征。方法：血浆样品经酸化后以乙醚- 环己烷（4 ∶ 1）提取,LC-MS/MS 分析,以Hedera ODS-2（150 mm×2.1 mm,5 μm）为分析柱,流动相为5 mmol?L-1 醋酸铵水溶液（含0.1% 乙酸）– 甲醇,梯度洗脱。二丙酸倍他米松、倍他米松及内标布地奈德的监测离子对分别为m /z 563.2（[M+CH3COO]-）→ 483.1、m /z 451.2（[M+CH3COO]-）→ 361.0 和m /z 489.3（[M+CH3COO]-）→ 357.1。对小型猪外用二丙酸倍他米松乳膏后其血浆中二丙酸倍他米松及代谢物倍他米松的浓度进行测定,并计算主要药动学参数。结果：二丙酸倍他米松血药浓度在26.85~644.4 ng?L-1 范围内线性关系良好,倍他米松血药浓度在10.62~637.2 ng?L-1 范围内线性关系良好。结论：本方法专属性强、简便、灵敏,可用于血浆样本中二丙酸倍他米松及其代谢物倍他米松的测定及药动学研究。     

液相串联质谱法同时定量检测生物样本中鞘脂类化合物

鞘脂类中的主要活性分子鞘氨醇1-磷酸（S1P）,可通过介导细胞增殖、分化和迁移等发挥广泛的生物学功能|同时,S1P可在相关酶的作用下转变为其它形式.本文旨在建立一种简便的鞘脂类的制备方法,并结合液相串联质谱（LC-MS/MS）快速检测生物样本中纳克水平的鞘脂类化合物. 采用甲醇沉淀或经典的脂质提取方法获得鞘脂类化合物,再采用LC-MS/MS在多反应监测模式（MRM）下进行定量分析. 实验结果表明,甲醇沉淀法可简便快捷地获得血浆或脂蛋白中鞘氨醇类化合物; S1P、二氢鞘氨醇（DH-S1P）和鞘氨醇（SPH）的定量限分别为110.5、215.6和44.3 pg;人血浆中S1P、DH-S1P和SPH的含量分别为257.8±49.4 nmol/L、93.5±17.3 nmol/L和44.6± 7.4 nmol/L, 鞘脂类化合物在人血浆脂蛋白上的分布存在显著性差异|在ApoE-/-小鼠血浆中S1P、DH-S1P和SPH的含量分别为590.1±78.2 nmol/L、197.8±60.6 nmol/L和35.4±16.7 nmol/L|每1×106个人脐静脉内皮细胞（EA.hy926）中,S1P和SPH的含量分别为103.7±21.8 pg和16.3±5.3 ng.甲醇沉淀法结合LC-MS/MS可简便快捷地对血浆和脂蛋白中的鞘脂类化合物进行定量检测. 该方法特别适用于大量生物样本中鞘脂类的快速定量分析.     

LC-MS/MS法测定苦参提取物中苦参碱和氧化苦参碱的质量浓度

摘要 目的：探讨液相色谱/串联质谱法(Liquid chromatography-tandem mass spectrometry,LC-MS/MS)测定苦参提取物中苦参碱和氧化苦参碱的质量浓度。方法：验证LC-MS/MS测定苦参提取物中苦参碱、氧化苦参碱的可行性与有效性,并判定检出限、重复性、稳定性、加样回收率。结果绘制苦参碱、氧化苦参碱的检出标准曲线。结果：LC-MS/MS检测苦参提取物中苦参碱与氧化苦参碱的一级质谱图和二级质谱图显示,苦参碱和氧化苦参碱的相对分子量为：248.36、246.36。色谱图显示苦参碱与氧化苦参碱无干扰色谱峰,各成分及内标峰形良好,表明该方法专属性强。在5 μg/L~500 μg/L浓度范围内,苦参碱、氧化苦参碱均呈现良好的线性关系,线性方程分别为y=6.10x-118.29和y=18.29x-14.22,检出限均为0.1 μg/L。LC-MS/MS法测定苦参提取物中苦参碱、氧化苦参碱的重复性、稳定性、加样回收率RSD均<5 %。结论：LC-MS/MS法测定苦参提取物中苦参碱和氧化苦参碱的质量浓度具有可行性、高灵敏度、高特异性等优点,可用于苦参提取物的质量控制与评价,并为后续他人的研究提供关键试验参考条件。     

检测小鼠肝炎病毒的SNaPshot分型技术及其应用

建立一种能对MHV_1、MHV_3、JHM、A_(59) 4种常见小鼠肝炎病毒(Murine Hepatitis Virus,MHV)进行分型检测的SNaPshot新方法。根据MHV 4种常见毒株基因序列比对结果,设计内外两对PCR通用引物和4个单碱基延伸引物,提取MHV 4种常见毒株RNA,逆转录后进行PCR扩增,纯化扩增产物,用SNaPshot方法进行单碱基延伸,将产物进行毛细管凝胶电泳,根据电泳结果分析毒株基因型。优化SNaPshot分析条件,进行灵敏度、特异性分析。用ELISA法和SNaPshot方法检测41例小鼠(Mus musculus)血清样本,将阳性样本进行克隆测序检测。当T1~T4引物修饰的poly T的数量分别为0、3、10和15,其浓度比为4︰6︰5︰10,引物大小分别为16 bp、19 bp、26 bp和31 bp时,SNa Phot分型检测MHV c DNA的最低浓度为1.25 mg/L,特异性为100%,与ELISA和T-克隆测序比较,其准确性为100%(41/41),阳性样本均为JHM毒株。实验结果说明,所建立的SNaPshot检测方法能对MHV_1、MHV_3、JHM、A_(59)进行分型检测,并且具有灵敏、特异、准确的优点。     

液相色谱-串联质谱法同时测定大鼠血浆中阿霉素和塞来昔布

目的:建立同时测定大鼠血浆中阿霉素和塞来昔布的液相色谱-串联质谱(LC/MS/MS)方法,研究这两种药物联合应用的药代动力学.方法:大鼠尾静脉注射阿霉素和塞来昔布,眼眶取血并抗凝,离心分离血浆,采用乙酸乙酯提取血浆中的阿霉素和塞来昔布,N2吹干乙酸乙酯,残留物用50μL甲醇溶解,取20μL用于LC/MS/MS分析.结果:用LC/MS/MS法检测大鼠血浆中阿霉素和塞来昔布的线性范围为1-800ng/mL,日内、日间精密度(RSD)均小于15%,检测血浆低、中、高三个浓度(8、50、500ng/mL)阿霉素的回收率分别为101.2%、95.1%和91.4%,检测血浆低、中、高三个浓度(8、50、500ng/mL)塞来昔布的回收率分别为105.6%、106.8%和93.7%.大鼠尾静脉注射5.8mg/kg阿霉素和3.8mg/kg塞来昔布的半衰期分别为2.3 h和3.6h,曲线下面积分别为670 ng·h·mL-1和1480ng·h·mL-1.结论:建立的方法灵敏、准确、快速,适甩于阿霉素和塞来昔布的药代动力学研究.     

不同放散试验对新生儿ABO溶血病的诊断价值对比

摘要 目的：分析与比较不同放散试验对新生儿ABO溶血病的诊断价值。方法：选择2017年9月至2019年6月在本院进行ABO溶血病检测的新生儿240例,取所有新生儿的静脉血样本2~3 mL,采用冷冻复融放散试验方法与改良热放散试验方法检测新生儿ABO溶血病的发生情况,并比较单独诊断和联合诊断的价值。结果：在240份标本中,冷冻复融放散试验检出新生儿ABO溶血病阳性130例,阳性检出率为54.2 %;改良热放散试验检出新生儿ABO溶血病阳性94例,阳性检出率为39.2 %;二者联合检出新生儿ABO溶血病阳性100例,阳性检出率为41.67 %,联合检出新生儿ABO溶血病阳性率和冷冻复融放散试验检出新生儿ABO溶血病阳性率显著高于改良热放散试验检出新生儿ABO溶血病阳性率(P<0.05)。临床最终诊断为新生儿ABO溶血病101例,阳性率为42.08 %,患儿ABO血型包括A型56例,B型45例。冷冻复融放散试验诊断新生儿ABO溶血病的敏感性和特异性为73.8 %和95.5 % ,ROC曲线面积0.775;改良热放散试验检诊断为新生儿ABO溶血病的敏感性和特异性为100 %和95.2 %,ROC曲线面积0.853;二者联合诊断对新生儿ABO溶血病的敏感性和特异性为90.0 %和97.85 %,ROC曲线面积0.872,联合诊断特异性优于改良热放散试验检诊和冷冻复融放散试验诊断,且改良热放散试验检诊敏感性优于冷冻复融放散试验诊断。结论：相对于冷冻复融放散试验,改良热放散试验对新生儿ABO溶血病的诊断敏感性更高,且不影响诊断特异性,两种放散方法联合检测具有更好的应用价值。     

基于不同浓度变量的温带落叶阔叶林CO2储存通量的误差分析

利用帽儿山温带落叶阔叶林通量塔8层CO₂/H₂O浓度廓线的测定数据,比较分析了基于不同浓度变量\[密度(ρ_c)、摩尔分数(c_c)和混合比(χ_c)\]计算CO₂储存通量(F_s)的误差.结果表明： 通量观测的控制体积内部干空气储存量不为常数,其波动可引起CO₂分子进出控制体积,即干空气储存通量调整项(F_sd)的变化.在夜间以及昼夜转换期,F_sd相对于涡动通量而言较大,忽略F_sd将为森林与大气之间净CO₂交换量的计算带来误差.大气水热过程对F_s计算引起的误差包括3方面：空气温度变化引起的误差最大,比大气压强(P)的影响高1个数量级;水蒸气的影响在温暖湿润的夏季大于P的影响,但在寒冷干燥的冬季则相反; P的效应在全年均较低.基于ρ_c、c_c和χ_c计算F_s分别平均高估CO₂有效储存通量(F_{s_E})8.5%、0.6%和0.1%.在通量计算过程中,建议选择对大气水热过程守恒的χ_c计算F_s.     

基于多次重复液相质谱生物实验数据校准方法研究

在蛋白质组学中,进行液相质谱(LC-MS)实验谱数据处理,发现并分析生物标志物的复杂肽或蛋白质样本的差异是重点,而校准相同样本的多次重复实验中肽链产生的洗脱时间峰信号(LC峰)是进行量化、分析差异的关键。目前多个重复实验数据的校准通常是在重复的实验数据集中根据液相二级质谱(LC-MS/MS)实验标识LC峰的时间特征,然后使用翘曲函数对时间特征进行对齐。由于多重数据的洗脱时间误差产生是随机的,统一使用翘曲函数校准会产生较大误差。为了解决这个问题,本研究重点研究了多个重复实验数据中LC峰的时间校准算法。我们选取了两个重复实验数据,采用机器学习的思路,通过选用两个数据的LC-MS/MS中重复检测到的肽链数据作为可信数据,部分选为训练序列,部分作为测试序列,建立统计数学模型,提出了一种新的校准算法,并采用测试序列对该统计模型进行准确率测试,表明算法的准确性达到95%以上;然后,将该模型应用在两个实验数据的所有LC-MS/MS肽链检测值上,提高检测值在多个数据中的覆盖率,表明覆盖率可以到达85%以上。     

EAHFE – TROPICA2 study. Prognostic value of troponin in patients with acute heart failure treated in Spanish hospital emergency departments

Objective: Evaluate the use of different cardiac troponin (cTn) immunoassays and the prognostic value of increased cTn values in patients diagnosed with acute heart failure (AHF) in the emergency department (ED).

Method: The epidemiology acute heart failure emergency-TROPonin in acute heart failure2 (EAHFE-TROPICA2) is a retrospective study including patients with AHF admitted in 34 Spanish EDs with cTn values determined in the ED. We studied the prevalence of elevated troponin (value above the established reference limit) for the different types of troponin. We also assessed crude and adjusted primary (1-year all-cause death) and secondary (30 d ED revisit due to AHF) outcomes for every type of cTn and different magnitudes of troponin elevation.

Results: We analysed 4705 episodes of AHF. Troponin was elevated in 48.4% of the cases (25.3% in cTnI, 37.9% in cTnT and 82.2% in hs-cTnT). Mortality at one year was higher in patients with elevated troponin (adjusted HR 1.61; CI 95% 1.38–1.88) regardless of the type of cTn determined. Elevated troponin was not related to ED revisit within 30 d after discharge (1.01; 0.87–1.19).

Conclusions: The use of conventional troponin in the ED is useful to predict one-year mortality in patients with AHF. Highly sensitive cTnT (hs-cTnT) elevations less than double the reference value have no impact on patient outcome.     

我国疾病靶点研究最新进展

主要通过对中国学者2013—2014 年间在国内外发表的相关论文进行查阅和整理,分类综述我国在神经退行性疾病、抑郁症、 心脑血管疾病、代谢性疾病、感染性疾病、肿瘤、自身免疫性疾病等各种重大疾病靶点研究方面的最新进展。     

药物作用靶点研究最新进展

通过对我国学者近2年在国内外发表的相关论文进行检索和整理,分类综述针对神经退行性疾病（如阿尔茨海默病、帕金森病等）、心血管疾病（如高血压、心律失常、心衰、冠心病、心肌梗死、动脉粥样硬化等）、脑血管疾病、代谢类疾病（如肥胖症、血脂异常、脂肪肝、糖尿病等）、感染性疾病（如艾滋病、流感、结核病等）、恶性肿瘤、自身免疫性疾病等多种疾病的药物作用靶点研究最新进展。     

基于疾病本体的疾病相似性计算方法

疾病相似性研究对于复杂疾病发病机制的理解、诊断、预测和药物研发具有重要意义.最近,研究人员通过集成多种疾病术语库,构建了描述疾病关系的疾病本体(disease ontology,DO),这为从DO角度研究疾病相似性打下了基础.本文综述了基于DO及其注释信息的疾病相似性计算方法,探讨了疾病相似性计算存在的问题和挑战,为疾病相似性进一步的研究提供有益参考.     

Mitochondria, calcium and cell death: A deadly triad in neurodegeneration

Mitochondrial Ca²⁺ accumulation is a tightly controlled process, in turn regulating functions as diverse as aerobic metabolism and induction of cell death. The link between Ca²⁺ (dys)regulation, mitochondria and cellular derangement is particularly evident in neurodegenerative disorders, in which genetic models and environmental factors allowed to identify common traits in the pathogenic routes. We will here summarize: i) the current view of mechanisms and functions of mitochondrial Ca²⁺ homeostasis, ii) the basic principles of organelle Ca²⁺ transport, iii) the role of Ca²⁺ in neuronal cell death, and iv) the new information on the pathogenesis of Alzheimer's, Huntington's and Parkinson's diseases, highlighting the role of Ca²⁺ and mitochondria.     

Clinical and Molecular Characteristics of Japanese Gaucher Disease

Clinical signs and symptoms of Gaucher disease are more severe in Japanese than in Jewish and other non-Japanese patients. A higher percentage of bone crises and splenectomy was demonstrated by Japanese patients, and there were five fatalities among patients with type 1 Gaucher disease. Additionally, neonatal Gaucher disease, clinically characterized by hydrops foetalis, was observed. Japanese patients with type 2 and type 3 disease also demonstrate clinical heterogeneity. About 100 alleles of patients with Japanese Gaucher disease were examined for genotype determination with the PCR and SSCP methods. About 18 different mutations, including several novel mutations in Japanese patients, were identified. The most common mutations in Japanese patients were 1448C(L444P), accounting for 41 (41%) of alleles. The second most prevalent mutation was 754A(F2131), accounting for 14 (14%) of alleles. Other alleles identified included the 1324C, IVS2 and other mutations. Unidentified alleles comprised 16% of the total number of alleles studied. To date, neither the 1226G (N370S) nor the 84GG mutation has been identified in the Japanese population, although these mutations account for about 70% and 10% of the mutations in Jewish and other non-Japanese populations, respectively. The phenotype-genotype correlation in Japanese patients is more complex compared with that of the Jewish population. In Japanese patients, the 1448C mutation, in either heteroallelic or homoallelic forms, exhibits both neurological and non-neurological phenotypes. Japanese patients with the 754A mutation also exhibit both neuronopathic and non-neuronopathic disease. On the other hand, patients with the D409H mutation show only type 3 neurological disease, and those with the 1447–1466 del 20 ins TG mutation have the severe, neonatal neurological form of Gaucher disease. The 1503T allele was present only in patients with type 1 non-neurological disease. However, since this correlation was observed only in young patients, we do not as yet know the final phenotypic outcome of this mutation. Probably, Japanese patients with Gaucher disease have few mutations that exhibit non-neurological signs and symptoms.     

Mitochondrial Dysfunction in Neurodegeneration

Numerous toxins are known to interfere with mitochondrial respiratory chain function. Use has been made of these in the development of pesticides and herbicides, and accidental use in man has led to the development of animal models for human disease. The propensity for mitochondrial toxins to induce neuronal cell death may well reflect not only their metabolic pathways but also the sensitivity of neurons to inhibition of oxidative phosphorylation. Thus, the accidental exposure of humans to l-methyl-4-phenyl-l,2,3,6-tetrahydropyridine and to 3-nitropropionic acid has led to primate models of Parkinson's disease and Huntington's disease, respectively. These models were made all the more remarkable when identical biochemical deficiencies were identified in relevant areas of humans suffering from the respective idiopathic diseases. The place of complex I deficiency in Parkinson's disease remains undetermined, but there is recent evidence to suggest that, in some cases at least, it may play a primary role. The complex II/III deficiency in Huntington's disease is likely to be secondary and induced by other pathogenetic factors. The potential to intervene in the cascade of reactions involving mitochondrial dysfunction and cell death offers prospects for the development of new treatment strategies either for neuroprotection in prophylaxis or rescue.     

Wildlife disease elimination and density dependence

Disease control by managers is a crucial response to emerging wildlife epidemics, yet the means of control may be limited by the method of disease transmission. In particular, it is widely held that population reduction, while effective for controlling diseases that are subject to density-dependent (DD) transmission, is ineffective for controlling diseases that are subject to frequency-dependent (FD) transmission. We investigate control for horizontally transmitted diseases with FD transmission where the control is via culling or harvest that is non-selective with respect to infection and the population can compensate through DD recruitment or survival. Using a mathematical model, we show that culling or harvesting can eradicate the disease, even when transmission dynamics are FD. Eradication can be achieved under FD transmission when DD birth or recruitment induces compensatory growth of new, healthy individuals, which has the net effect of reducing disease prevalence by dilution. We also show that if harvest is used simultaneously with vaccination, and there is high enough transmission coefficient, application of both controls may be less efficient than vaccination alone. We illustrate the effects of these control approaches on disease prevalence for chronic wasting disease in deer where the disease is transmitted directly among deer and through the environment.     

Soluble CD40 ligand in haemodialysis patients: survival impact and cardiovascular prognostic role

Context: Soluble CD40 ligand (sCD40l) can predict cardiovascular events (CVE) and mortality in haemodialysis (HD) patients (short-, medium-term follow-up studies).

Objective: To evaluate the relationship between sCD40l and survival, CVE and mortality in HD patients on long-term follow-up.

Methods: We registered 46?HD patients’ baseline characteristics, mortality and CVE for 108 months.

Results: SCD40l correlated positively with C-reactive protein, was higher in survivors, but had no impact on survival and was not predictive for CVE or CV mortality.

Conclusion: The levels of sCD40l have no influence on survival or CVE and mortality in HD patients in a long-term follow-up.     

More than metabolic: Considering the broader paleoepidemiological impact of vitamin D deficiency in bioarchaeology

Vitamin D deficiency has traditionally been viewed as a metabolic bone disease by bioarchaeologists and considered primarily in terms of the development of specific musculoskeletal changes used for diagnosis in paleopathological research. These skeletal manifestations are usually interpreted as representing general ill‐health. Clinical research shows that vitamin D is also integral to a number of extra‐skeletal physiological processes including immunoregulation, blood pressure homeostasis, cell division, and programmed cell death. Vitamin D deficiency and sub‐clinical insufficiency are thought to be risk factors for infectious and autoimmune diseases, as well as certain cancers and cardiovascular diseases. Epidemiological work indicates that the skeletal manifestations of vitamin D deficiency represent the extreme end of a spectrum of morbidity associated with negative health outcomes, including increased risk for secondary tuberculosis. This article provides a review of clinical research on the extra‐skeletal roles of vitamin D and the pathological consequences of poor vitamin D status. Additionally, it presents an interpretive model for bioarchaeological analyses of rickets and osteomalacia for consideration of the whole‐body impact of poor vitamin D nutriture and possible comorbidities that may have affected the wider population. Am J Phys Anthropol 160:183–196, 2016. © 2016 Wiley Periodicals, Inc.