帕金森病相关血清蛋白质标志物的筛选及鉴定

 目的：探讨并初步鉴定帕金森病患者血清中相关蛋白质作为特异标志物的可能性。方法：应用表面增强激光解吸电离飞行时间质谱(SELDI-TOF-MS)结合纳米磁珠技术检测44例帕金森病和60例健康对照的血清标本,应用生物信息学方法筛选差异蛋白峰,经高效液相色谱（HPLC）分离出差异蛋白,酶解后进行液质联用串联质谱（LC-MS/MS）分析,利用Xcalibur的程序组件BioWorks 3.2完成蛋白质序列数据库鉴定分析。结果：经SELDI-TOF-MS结合纳米磁珠技术筛选出质荷比m/z位于8 937的蛋白质在帕金森病中高表达(帕金森病组表达强度为27.47±16.58,正常组表达强度为5.01±3.47),有显著差异(P<0.01);6 636和8 697的蛋白质在帕金森病中低表达(帕金森病组表达强度为5.43±2.66和20.22±9.57,正常组表达强度为18.85±7.56和51.13±26.22), 有显著差异(P<0.01)。联合上述3种潜在蛋白质标志物,可区分帕金森病组和对照组,其中帕金森病患者检出率为90.0%(27/30),健康者检出率为92.5%（37/40）。对m/z为6 636、8 697和8 937的标志物进行鉴定,结果分别为载脂蛋白C-I、载脂蛋白 C-III 和补体成分3a。结论：鉴定出的载脂蛋白 C-I、载脂蛋白C-III和补体成分3a在帕金森病的诊断中具有一定价值,值得进一步研究和探讨。     

Identification of pathogenic microorganisms directly from positive blood vials by matrix‐assisted laser desorption/ionization time of flight mass spectrometry

Matrix‐assisted laser desorption/ionization time of flight mass spectrometry (MALDI‐TOF MS) is a promising and fast method for identifying fungi and bacteria directly from positive blood cultures. Various pre‐treatment methods for MALDI‐TOF MS identification have been reported for this purpose. In‐house results for identification of bacterial colonies by MALDI‐TOF MS using a cut‐off score of 1.5 did not reduce the diagnostic accuracy compared with the recommended cut‐off score of 1.8. A 3‐month consecutive study of positive blood cultures was carried out in our laboratory to evaluate whether the Sepsityper? Kit (Bruker Daltonics) with Biotyper 2.0 software could be used as a fast diagnostic tool for bacteria and fungi and whether a 1.5 cut‐off score could improve species identification compared with the recommended score of 1.8. Two hundred and fifty‐six positive blood vials from 210 patients and 19 blood vials spiked with fungi were examined. Using the cut‐off score of 1.8, 81% Gram‐negative bacteria were identified to the species level compared to 84% using a cut‐off score of 1.5. For Gram‐positive bacteria 44% were identified to the species level with a cut‐off of 1.8 compared to 55% with the value of 1.5. The overall identification rate was 63% (cut‐off 1.5) and 54% (cut‐off 1.8). Seventy‐seven per cent of fungal species were identified with both log scores. MALDI‐TOF MS was in this study found to be a powerful tool in fast diagnosis of Gram‐negative bacteria and fungi and to a lesser degree of Gram positives. Using 1.5 as cut‐off score increased the diagnosis for both Gram‐positives and ‐negatives bacteria.     

Tea catechin auto-oxidation dimers are accumulated and retained by Caco-2 human intestinal cells

Despite the presence of bioactive catechin B-ring auto-oxidation dimers in tea, little is known regarding their absorption in humans. Our hypothesis for this research is that catechin auto-oxidation dimers are present in teas and are absorbable by human intestinal epithelial cells. Dimers (theasinensins [THSNs] and P-2 analogs) were quantified in commercial teas by high-performance liquid chromatography-mass spectrometry. (−)-Epigallocatechin (EGC) and (−)-epigallocatechin gallate (EGCG) homodimers were present at 10 to 43 and 0 to 62 μmol/g leaf, respectively. The EGC-EGCG heterodimers were present at 0 to 79 μmol/g. The potential intestinal absorption of these dimers was assessed using Caco-2 intestinal cells. Catechin monomers and dimers were detected in cells exposed to media containing monomers and preformed dimers. Accumulation of dimers was significantly greater than monomers from test media. Three-hour accumulation of EGC and EGCG was 0.19% to 0.55% and 1.24% to 1.35%, respectively. Comparatively, 3-hour accumulation of the EGC P-2 analog and THSNs C/E was 0.89% ± 0.28% and 1.53% ± 0.36%, respectively. Accumulation of P-2 and THSNs A/D was 6.93% ± 2.1% and 10.1% ± 3.6%, respectively. The EGCG-EGC heterodimer P-2 analog and THSN B 3-hour accumulation was 4.87% ± 2.2% and 4.65% ± 2.8%, respectively. One-hour retention of P-2 and THSNs A/D was 171% ± 22% and 29.6% ± 9.3% of accumulated amount, respectively, suggesting intracellular oxidative conversion of THSNs to P-2. These data suggest that catechin dimers present in the gut lumen may be readily absorbed by intestinal epithelium.     

Characterization of New Amphiphilic Block Copolymers of N‐Vinyl Pyrrolidone and Vinyl Acetate, 1 – Analysis of Copolymer Composition,End Groups,Molar Masses and Molar Mass Distributions

New amphiphilic block copolymers consisting of N‐vinyl pyrrolidone and vinyl acetate were synthesized via controlled radical polymerization using a reversible addition/fragmentation chain transfer (RAFT)/macromolecular design via the interchange of xanthates (MADIX) system. The synthesis was carried out in 1,4‐dioxane as process solvent. In order to get conclusions on the mechanism of the polymerization the molecular structure of formed copolymers was analysed by means of different analytical techniques. ¹³C NMR spectroscopy was used for the determination of the monomer ratios. End groups were analysed by means of matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry. This technique was also used to determine possible fragmentations of the RAFT end groups. By means of a combination of size exclusion chromatography, ¹³C NMR and static light scattering molar mass distributions and absolute molar masses could be analysed. The results clearly show a non‐ideal RAFT mechanism.

     

失血性休克大鼠血浆蛋白质的差异分析

目的 应用蛋白质双向凝胶电泳(two-dimensional polyacrylamide gel electrophoresis, 2-DE) 技术分析在急性重度失血性休克(acute severe hemorrhagic shock, ASHS)条件下大鼠血浆蛋白质组表达的差异.方法 16 只雄性 Wistar大鼠随机分成对照组(sham hemorrhage shock, SHS)和ASHS模型组,每组8只.采用股动脉放血的方法制备模型,在规定时间内处死大鼠并收集血浆,去除血浆中的高丰度蛋白后进行 2-DE,运用 Image Master 2D Platinum v 5.0 凝胶图像分析软件对 2-DE 凝胶图像进行差异表达分析,有意义的差异蛋白质点用基质辅助激光解析电离飞行时间质谱进行肽质量指纹图谱分析,借助Swiss-prot 数据库进行蛋白质搜索和鉴定.结果 SHS 组和 ASHS 组肝脏的 2-DE 图谱分别平均识别到(570.0±0.9)和(578.0±1.4)个蛋白质点,SHS 组和 ASHS 组肝脏间平均匹配率达 83.6 %～86.2 %,共发现 8 个差异有意义的蛋白质点,鉴定出了白蛋白、甲状腺激素结合蛋白-D 链蛋白和信号素-3D 前体等 3 种蛋白质.结论 以 2-DE 技术得到重复性和分辨率都较好的 2-DE 图谱,并初步鉴定急性重度失血性休克后大鼠血浆去除高丰度蛋白后的差异表达蛋白质,为深入研究失血性休克的生理病理机制及寻找失血性休克预防和治疗的生物标志物提供了依据.     

Safety assessment of a novel ingredient for removable chewing gum

Rev7™ is an indigestible gum polymer used for the manufacturing of chewing gum. It allows for the formulation of chewing gum with low adhesion; thus can be readily removed from surfaces such as sidewalks, clothing, carpets and furniture. In a toxicological safety assessment, Rev7™ was found to be non-mutagenic in the AMES assay. The highest concentration tested in a mouse lymphoma thymidine kinase locus gene mutation assay induced a slight but biologically relevant increase in mutations under non-metabolic activation conditions after 24 h. Because of this finding, a mouse micronucleus assay was performed, and the test article was found to be negative for inducing chromosomal damage. A 28-day repeated oral toxicity study resulted in a NOAEL of 80,000 ppm; the highest concentration tested. Rev7™ was found to be free from contaminants such as heavy metals, monomers, and solvents. Lastly, Rev7™ did not demonstrate skin-sensitizing properties in the murine local lymph node assay.     

Nitrosamine Contamination in Pharmaceuticals: Threat,Impact, and Control

Nitrosamine-contaminated medicinal products have raised safety concerns towards the use of various drugs, not only valsartan and all tetrazole-containing angiotensin II receptor blockers, but also ranitidine, metformin, and other medicines, many of which have been recalled and prone to shortage. At any stages, from drug substance synthesis throughout each product's lifetime, these impurities may evolve if an amine reacts with a nitrosating agent coexisting under appropriate conditions. Consequently, drug regulatory authorities worldwide have established stringent guidelines on nitrosamine contamination for all drug products in the market. This review encompasses various critical elements contributing to successful control measures against current and upcoming nitrosamine issues, ranging from accumulated knowledge of their toxicity concerns and potential root causes, precise risk evaluation, as well as suitable analytical techniques with sufficient sensitivity for impurity determination. With all these tools equipped, the impact of nitrosamine contamination in pharmaceuticals should be mitigated. An evaluation aid to tackle challenges in risk identification, as well as suitable industry-friendly analytical techniques to determine nitrosamines and other mutagenic impurities, are among unmet needs that will significantly simplify the risk assessment process.     

液相色谱-串联质谱法测定人血浆中米格列奈浓度及其药动学研究

目的建立一种快速分析测定人血浆中米格列奈的液相色谱-串联质谱色谱法,用以研究米格列奈在健康人体内的药动学。方法以那格列胺为内标,血浆酸化后经液液萃取后,采用液相色谱-串联质谱法以多反应检测方式进行测定,选择监测的离子为m/z 316.2→298.2(米格列奈)和m/z 318.2→120.2 (那格列胺)。流动相以甲醇-10 mmol·L~(-1)醋酸铵水溶液(75:25,V/V),流速0.3 mL·min~(-1),色谱柱为Agilent Zorbax Eclipse Plus C_(18)(1.8μm,3 mm×150 mm);柱温:30℃。结果米格列奈在0.502 0～4 016μg·L~(-1)浓度范围内呈良好的线性关系(r=0.995),最低检测浓度为0.502 0μg·L~(-1),精密度和准确度试验均符合生物分析要求,应用此法测得5、10、20 mg剂量组不同给药方式、多个时间点的米格列奈血药浓度,结果呈线性动力学特征。结论该方法灵敏度高、专属性强、准确、简便,适用于米格列奈的人体药动学研究。     

Diagnosis of tumor-like polypoid lesions of gallbladder by serum proteomic fingerprint

Objective:To search the specific serum proteins in tumor-like polypoid lesions of the gallbladder(PLG)patients. Methods:Surface enhanced laser desorption/ionization time of flight mass spectrometry (SELDI-TOF-MS)technique and WCX Magnetic Beads were used to detect the serum proteomic fingerprint of 23 patients with tumor-like PLG,21 patients with non tumor-like PLG and 26 normal persons.Biomarker Wizard and Biomarker Patterns Software were used in combination to analyze the data.Results:In the preliminar...     

液相色谱-串联质谱法测定辛伐他汀片的人体药动学和生物等效性

目的:建立人血浆中辛伐他汀浓度的液相色谱-串联质谱(LC-MS／MS)测定法,研究健康受试者单剂量口服辛伐他汀受试或参比制剂后的药动学和生物等效性。方法:20名健康男性受试者进行随机双交叉试验,分别单剂量口服20 mg辛伐他汀受试制剂和参比制剂,采用LC-MS／MS以洛伐他汀为内标正离子选择性反应检测测定辛伐他汀血浆浓度,计算两者的药动学参数及相对生物利用度。结果:受试制剂和参比制剂的血药浓度水平一致,主要药动学参数如下:c_(max)分别为(8．0±s 1．9)和(8．1±1．8)μg·L~(-1);t_(max)分别为(1．9±0．3)和(1．9±0．3)h;t_(1/2)分别为(3．7±1．4)和(4．1±2．2)h;AUC_(0～24)分别为(30±8)和(30±6)μg·h·L~(-1);AUC`(0～∞)分别为(30±8)和(31±7)μg·h·L~(-1)。由2种制剂的AUC_(0～24)计算,受试制剂的相对生物利用度为(101±20)%。结论:建立的LC-MS／MS测定法专属、准确、灵敏度适宜。测得辛伐他汀受试制剂和参比制剂生物等效。