冠心病患者糖化血红蛋白水平检测对评估患者冠状动脉病变程度的价值探究

目的通过观察了解糖化血红蛋白HbA1c水平与冠状动脉病变严重程度的相关关系。方法随机选取2013年1月—2015年1月来遵义医学院第五附属(珠海)医院心血管内科住院并进行选择性冠脉造影(CAG)的96例患者,同时对这96例患者进行糖化血红蛋白测定,回顾性分析冠心病患者的临床症状、体征、诊疗等相关指标,以了解HbA1c水平与冠状动脉病变程度的关系。结果对冠心病患者进行临床调查,并对结果进行记录,冠心病组和对照组HbA1c水平比较可知,冠心病组HbA1c水平高于对照组(P0.05)。结论对冠心病患者进行糖化血红蛋白水平检测,严格控制高血糖,降低HbA1c水平,通过改善血管内皮细胞功能,对延缓或阻止动脉粥样硬化,减少心血管并发症和后遗症的发生有着重要意义。     

糖化蛋白质的电化学检测及其临床应用

糖化血红蛋白(HbA1c)是血液中葡萄糖分子与血红蛋白分子的β-链末端氨基发生非酶反应的产物 [1-2].血液中葡萄糖的浓度越大,红血球中HbA1c的含量就越高,因此红血球中HbA1c含量高低反映了血液中葡萄糖的水平.由于人体内红血球的寿命大约为100～120 d,因此临床上测定HbA1c含量可提前2～3个月预知体内葡萄糖水平,对糖尿病患者血糖水平的中长期控制及糖尿病的早期诊断具有重要的实践意义[3,4].通常HbA1c的含量用HbA1c占血红蛋白总量的百分比来表示,临床上HbA1c含量的参考值为5%～20%,并认为4%～6%是正常的.目前临床上有多种方法用来测定HbA1c含量,比如免疫、离子交换色谱、硼亲和色谱以及电泳等[5-8]. 这些方法在光度定量测定HbA1c含量之前均包含有1个分离步骤,例如基于HbA1c与Hb表面电荷差别的离子交换色谱和电泳分离以及基于HbA1c与Hb结构差别的免疫和硼亲和色谱方法.     

借2—（2—咪唑偶氮）—苯酚—4—磺酸螯合物和液相色谱分离铬,钼,钒和钴

研究了离子对反相高效液相色谱分离铬（Ⅱ）、钼（Ⅵ）、钒（Ⅳ）和钴（Ⅱ）之２－（咪唑偶氮）－苯酚－４－磺酸螯合物的柱前衍生条件；检测波长的选择；流动相中有机溶剂、四丁基溴化铵及酸度的影响，并讨论了１５种外来离子的干扰情况。本方法已用于合金钢和钒喳的分析。检测限为０．４ｎｇ铬、１．２ｎｇ钼和０．１ｎｇ钒。     

基于微流控芯片电泳化学发光检测人单个红细胞中血红蛋白的含量

运用自行设计组装的微流控芯片电泳化学发光检测装置和单细胞分析专用玻璃微流控芯片,建立了一种测定人单个血红细胞中血红蛋白(Hb)含量的新方法。该方法采用双T型的窄进样通道,宽反应通道及适中分离通道的玻璃微流控芯片,集成单个细胞的进样、固定、溶胞、分离和检测等操作于一块微流控芯片上。以p H 10.5的硼砂缓冲液为电泳介质,选用鲁米诺-过氧化氢化学发光体系,对人单个红细胞中血红蛋白的含量进行测定。血红蛋白的质量在2.0~90 pg范围内,与化学发光强度(峰高)呈良好的线性关系,检出限(S/N=3)为0.8 pg。通过对19个血红细胞进行检测,得到人单个血红细胞中血红蛋白的含量在14~68 pg范围内,该结果与无氰HGB测量法测得的总体细胞血红蛋白的平均值(34.5 pg)基本一致。     

阴离子交换树脂固相萃取分离全血中血红蛋白的研究

以330阴离子交换树脂为吸附材料建立了固相萃取分离血红蛋白的方法。利用血红蛋白和树脂之间的疏水作用力将血红蛋白吸附到树脂上,以Tris—HCl缓冲液（PH-8．9）为洗脱剂回收血红蛋白。考察了溶液PH值、吸附时间、离子强度、洗脱剂的种类及其酸度等对分离纯化效率的影响。在最优实验条件下,树脂对血红蛋白的吸附率和洗脱率分别为87％和70％,吸附容量为42．9μg／mg。吸收光谱和SDS—PAGE凝胶电泳证明,该方法可有效地从人全血中分离出纯度较高的血红蛋白。     

毛细管电泳-间接化学发光法分离检测儿茶酚胺及儿茶酚

根据儿茶酚胺及儿茶酚淬灭铁氰化钾-鲁米诺体系发光的原理,利用毛细管电泳．化学发光联用技术分离测定了3种儿茶酚胺和儿茶酚,并优化了检测和分离条件。在最佳条件下,测得多巴胺、肾上腺素、去甲肾上腺素和儿茶酚的检出限分别为0．33、1．8、2、4和0、12μmol／L。本方法具有一定的选择性,对于医用注射液及尿样在未经预处理条件下可直接进行分离分析,结果令人满意。     

反相高效液相色谱法同时测定人血浆中的西诺沙星和萘啶酸

提出了同时测定人血浆中的西诺沙星和萘啶酸的高效液相色谱方法。采用反相C18柱、254nm紫外检测,乙腈－甲醇－0．01mol/L草酸（30：5：65,V／V;pH4．55）体系,实现了二者的良好分离。分别考察了磷酸缓冲溶液和草酸缓冲溶液作为流动相弱溶剂,结果草酸缓冲溶液能有效改善峰形、降低检测限。在不同pH值（pH2．80,4．08,4．55）进行了实验,pH4．55获得最佳分离,最低检测量分别为0．14ng（西诺沙星）和0．24ng（萘啶酸）,比文献报道的同类药物最低检测量低10倍。     

二氧化钛结合超滤膜富集和分离肿瘤患者唾液中的磷酸化肽和唾液酸化糖肽

利用TiO2富集、超滤膜滤过实现了选择性富集和分离磷酸化肽和含唾液酸的N-链接糖肽.首先选取截留分子量为1×104的超滤膜分离糖肽和磷酸化肽,然后利用酪蛋白和牛血清白蛋白的酶解物验证所建立的方法,该方法的最低检出限是0.16 pmol.将上述方法应用于肺癌患者的唾液检测,成功检测并鉴定到8个磷酸化肽和5个糖肽.本方法可有效提高磷酸化肽和糖肽检测的选择性和灵敏度,为疾病生物标志物的检测提供了新方法.     

一步法和两步法毛细管等电聚焦测定蛋白质和多肽等电点的比较

利用一步法和两步法毛细管等电聚焦(cIEF)方法分离测定了蛋白质和多肽的等电点(pI)。讨论了两步法等电聚焦过程所需的溶液组成、样品进样体积、聚焦电压、聚焦时间和分离条件等因素对分离效果的影响。并对一步法和两步法进行了比较。对细胞色素C、血红蛋白、肌红蛋白、转铁蛋白和牛血清白蛋白以及6种多肽的分析结果表明:一步法步骤简单,分离速度快,可测定单一组分的pI,也能快速分离混合蛋白和多肽,但分离度较差,且不能同时准确测定各组分的pI;两步法步骤复杂,分析时间较长,但能够同时分离并准确测定混合样品中各组分的pI,所测的pI值与单一组分进行测定的结果基本一致。两种方法可相互结合、互为补充,可广泛应用于两性生物微粒等电点的快速和准确测定。     

离子交换色谱法同时测定啤酒中有机酸和无机阴离子

建立了用亲水性阴离子交换分离柱,KOH为淋洗液等浓度泵作梯度淋洗,电导检测,同时分离和检测16种无机阴离子和低分子量有机酸的离子色谱法。方法对所测无机阴离子和有机酸检出限在9．3～32μg/L之间;线性范围均在2个数量级以上;回收率在90．2％～107．2％之间。方法用于啤酒样品的分析,结果满意,样品的RSD小于5．3％(n=7)。     

Multiplex target protein imaging in tissue sections by mass spectrometry--TAMSIM

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-MS) is becoming a popular tool for imaging histological sections. Currently, this technology is used to image naturally occurring molecules. Here we report a novel development for multiplex imaging of candidate proteins. Rather than detecting whatever molecules happen to be present and above the detection threshold in the desorption pixel, we attach photocleavable mass tags to antibodies to target proteins. 'Staining' of histological sections is carried out similarly to common immunohistochemical procedures with chemiluminescent or fluorescent detection using all antibodies of a multiplex simultaneously. Mass tags with discrete masses are released from their respective antibodies by a laser pulse at 355 nm without added matrix. After scanning, mass spectrometry images are created for the mass of each tag. In contrast to fluorescent tags, mass tags do not exhibit mutual quenching. Sections of healthy human pancreatic tissue were imaged to visualize synaptophysin in neuroendocrine cells, and sections from human lymph node and liver invaded by metastatic melanoma to localize the cancer markers PS100 and HMB45 simultaneously. All these proteins are below the detection threshold of direct MALDI-MS imaging. This method is termed TAMSIM for TArgeted multiplex Mass Spectrometry IMaging.     

Coupling chemiluminescence with capillary electrophoresis to analyze single human red blood cells

In this paper, we describe a new method for determination of hemoglobin of single red blood cells by coupling chemiluminescence with capillary electrophoresis (CL-CE). The chemiluminescent detection is based on the catalytic effects of hemoglobin on the luminol-hydrogen peroxide reaction. The conditions of chemiluminescent reaction and capillary electrophoresis were investigated. Hemoglobin in human blood samples was detected with the present method, the linear range from 1.7 μg mL⁻¹ to 6.8 μg mL⁻¹ was tested, and the correlation coefficient of 0.997 and low detection limit of 0.17 μg mL⁻¹ (approximately 2.2 pg, S/N = 3) were obtained. Cell injection procedure was improved, and the method was successfully used to determine hemoglobin of single red blood cells and the statistical result of the average content of hemoglobin in 26 human red blood cells was 23.6 pg. Compared to other current methods, CE with CL system is simple, sensitive and will become an attractive alternative method for single cell analysis.     

The use of charge-coupled devices in the quantitative evaluation of images, on photographic film or membranes, obtained following electrophoretic separation of DNA fragments

Charge-coupled devices can provide an excellent means for the quantification of DNA band images on membranes and photographic film. However, proper consideration must be given to important parameters such as signal-to-noise ratio and resolution, especially in applications which demand the use of large format media. When employed in the direct imaging of chemiluminescent blots, the charge-coupled device can provide equal or better sensitivity than that obtained by indirect methods using film, with the additional advantages of wide dynamic range and freedom from the vagaries of film processing.     

Straightforward protein immobilization on Sylgard 184 PDMS microarray surface

In this work, a straightforward technique for protein immobilization on Sylgard 184 is described. The method consists of a direct transfer of dried protein/salt solutions to the PDMS interface during the polymer curing. Such non-conventional treatment of proteins was found to have no major negative consequence on their integrity. The mechanisms of this direct immobilization were investigated using a lysine modified dextran molecule as a model. Clear experimental results suggested that both chemical bounding and molding effect were implicated. As a proof of concept study, three different proteins were immobilized on a single microarray (Arachis hypogaea lectin, rabbit IgG, and human IgG) and used as antigens for capture of chemiluminescent immunoassays. The proteins were shown to be easily recognized by their specific antibodies, giving antibody detection limits in the fmol range.     

Chemiluminescent imaging analysis of interferon alpha in serum samples

Enzyme-linked immunosorbent assay (ELISA), horseradish peroxidase (HRP)-catalyzed fluorescent reaction, and oxalate chemiluminescence imaging analysis have been combined to develop a sensitive, simple, and rapid method for analysis of interferon alpha (α-IFN) in human serum samples. A typical “sandwich type” immunoassay was used. Reaction of o-phenylenediamine (OPD) with hydrogen peroxide (H₂O₂), catalyzed by HRP, produced 2,3-diaminophenazine (PDA), which was detected by chemiluminescence imaging analysis with the bis(2,4,6-trichlorophenyl)oxalate (TCPO)–H₂O₂–glyoxaline–PDA chemiluminescent system. The TCPO chemiluminescent imaging system is more sensitive and the chemiluminescence quantum yield is at least five times higher than for the luminol–H₂O₂–HRP–PIP (p-iodophenol) chemiluminescent imaging system. The results showed there was a very good linear correlation between response and amount of α-IFN in the range 1.3–156.0 pg mL⁻¹ (R = 0.9991) and the detection limit was 0.8 pg mL⁻¹ (S/N=3). The relative standard deviation (n = 9) was 4.7%. The proposed method has been used for successful analysis of the amount of α-IFN in human serum. The results obtained compared well with those obtained by conventional colorimetric ELISA and luminol chemiluminescent ELISA. Figure Procedures of the proposed method     

10-甲基吖啶苯酚酯衍生物的合成、表征及化学发光特性

吖啶-9-羧酸苯酚酯是一类效率较高的化学发光试剂.其结构一般有两部分组成,吖啶环发光部分和9-羧酸苯酚酯离去基团.本文对离去基团进行修饰,分别在离去基团苯酚部分的2,5或2,6位引入取代基CF₃、NO₂、Br、CH₃,合成了4个新吖啶酯衍生物,并对它们的分子结构进行了表征.4个新化合物和模型化合物(无取代基)比较,均表现出较好的化学发光效率;发光动力学基本符合文献报道规律.而且化学发光法对其水解稳定性考察显示,取代基的电性和位阻是影响稳定性的两大因素.     

A Glowing Trajectory between Bio‐ and Chemiluminescence: From Luciferin‐Based Probes to Triggerable Dioxetanes

Bioluminescent and chemiluminescent probes are widely used for noninvasive imaging applications because of their high sensitivity and the simplicity of the equipment required to perform the measurement. Synthetic luciferin‐analogue probes with in vivo imaging performance better than that of luciferin are now available. In addition, caged luciferin‐based bioluminogenic probes have been emerged as a general tool for the visualization of different enzymes and analytes in vivo. Recent discoveries have led to development of highly efficient chemiluminescent probes that are extremely bright under physiological conditions. As discussed in this Minireview, chemiluminescence is ready to realize its potential as a valuable tool for imaging in living systems.     

Development of a sensitive, rapid, biotin-streptavidin based chemiluminescent enzyme immunoassay for human thyroid stimulating hormone

A highly sensitive "two-site" chemiluminescent immunoassay specific for human thyroid stimulating hormone (TSH) was developed. The signal amplification was achieved via a biotin-streptavidin system (BSAS). The HRP-luminol-H(2)O(2) chemiluminescent system with high sensitivity was chosen as the detection system. Biotinylated anti-TSH monoclonal antibody (MAb) and HRP-labeled streptavidin were first synthesized. Then the signal amplification was achieved through the interaction between the biotinylated anti-TSH MAb and the HRP-streptavidin conjugate. The light intensity developed was in proportion to the TSH present in the samples. The assay showed little cross-reactivity with three other glycoprotein hormones (human chorionic gonadotropin (HCG), luteinizing hormone (LH), follicle stimulating hormone (FSH)) due to the high specificity of the antibody. The working range for human thyroid stimulating hormone was 0.1-40 mU L(-1). Both the intra-assay and inter-assay coefficients of variation were less than 10% for the BSAS based chemiluminescent enzyme immunoassay (CLEIA). The proposed assay had a sensitivity of 0.01 mU L(-1) which was 10-fold higher than the HRP-MAb conjugate based TSH immunoassay. Thus the higher sensitivity facilitated the clinical testing for thyroid states. The effects of several reaction parameters, such as incubation time, temperature, and reaction volume of the method, were also studied. This method has been successfully applied to the evaluation of TSH in human serum. Compared with the commercial enzyme chemiluminescent immunoassay, the correlation was satisfied.     

Direct chemiluminescent immunodetection of proteins in agarose gels

Chemiluminescent immunodetection of proteins separated by polyacrylamide gel electrophoresis is generally performed only after Western blotting. Agarose gels are adequately permeable to allow immunoprobing directly in the gel. Chemiluminescent substrates had not been applied for direct immunoprobing of agarose gels. In a comparison with direct immunostaining of fibrinogen derivatives with horse radish peroxidase (HRP)-conjugated primary antibody using 3,3'-diaminobenzidene (DAB) yielding a sensitivity in the low nanogram range, a luminol-based chemiluminescent detection extended sensitivity to the mid-picogram range with seemingly no interference from either regular or glyoxyl agarose gels. The high sensitivity of chemiluminescence extends utility of direct immunoprobing of either agarose or glyoxyl agarose composite gels for detection and measurement of both high and low molecular weight proteins/peptides which are not easily detected/measured by Western blotting. However, due to the thickness of the gels, direct immunoprobing can be quite laborious. To eliminate that drawback, we describe a simplified approach, converting the thick gels to thin ones prior to probing, that makes direct immunoprobing as easy as Western blotting.     

Development of an electrophoretic method based on nanostructured materials for HbA1c determination

Glycosylated hemoglobin (HbA1c) detection is performed routinely in hospitals as it is the most widespread confirmatory diagnosis of diabetes mellitus. Here we present a novel CE method for measuring HbA1c by introducing silica nanoparticles (NPs) modified with a boronic acid derivative (sugar loadings of 51 ± 2 μg/mg) as pseudo‐stationary phase. Before the sample injection, SiO₂NP─B(OH)₂ were introduced via pressure. Electrophoretic separation was explored through variation of the buffer pH and separation voltage, being the best separation, resolution and shorter separation time achieved with a 25 mM phosphate buffer pH 6.5. The calibration curve obtained was expressed as Area = 182.05%⁻¹ × HbA1c − 377.02; R² = 0.9826, using a UV/VIS absorbance detector at 415 nm (diode array). No interferences were observed from carbamylated or acetylated hemoglobin and the method shows a noteworthy stability. A paired t‐test was applied to compare the developed CE method with a commercial HbA1c test and no significant variations have been observed at a 90% significance level.