氧化剂对血痕的金标快速检测法影响的研究

金标快速检测法是一种能够对血痕中血液是否为人血进行快速种属认定的检测方法,但血痕中氧化剂的存在会使人血中的血红蛋白(Hb)氧化成高铁血红蛋白(MetHb),失去载氧活性,从而导致胶体金检测试纸条(FOB)种属检测不能检测出人血的存在。本文探究氧化剂的反应机理和血红蛋白被氧化的机理,并通过实验,获得导致胶体金检测试纸条失效的氧化剂最低剂量。     

氮氧自由基定点修饰牛血红蛋白的制备和抗自氧化研究

摘要:以4-（N-马来酰亚胺基）-2,2,6,6-四甲基哌啶-1-氮氧自由基( Mal-Tempol) 与牛血红蛋白（Hb）为原料,制备了氮氧自由基定点修饰在巯基上的血红蛋白( Mal-Tempol-Hb)。利用分光光度法测定了氮氧自由基的修饰度,通过紫外光谱研究了有无叠氮化钠（NaN3）介导下,Hb和Mal-Tempol-Hb中高铁血红蛋白(MetHb)含量随时间变化的过程。结果表明,无介导下,3h时Mal-Tempol-Hb中MetHb的摩尔百分含量由修饰前的44%下降为修饰后的28%;在NaN3介导下,3h时Mal-Tempol-Hb中MetHb的摩尔百分含量从修饰前的50%下降为修饰后的32%。氮氧自由基的修饰有效地降低了血红蛋白的自氧化速率。     

阿魏酸修饰的血红蛋白自氧化的研究

为研究FA-Hb的自氧化,以阿魏酸酯(FA-NBA)与血红蛋白(Hb)为原料,制备了阿魏酸血红蛋白(FA-Hb)。通过紫外光谱扫描、血氧分析仪分析了修饰前后血红蛋白的特征吸收峰的变化和氧饱和曲线的差别;研究了有无叠氮钠、黄嘌呤-黄嘌呤氧化酶介导下,血红蛋白和阿魏酸修饰的血红蛋白的自氧化速率的改变。结果表明,阿魏酸修饰血红蛋白,保持了血红蛋白的功能结构,仍具备血红蛋白的载氧活性,有效降低了自氧化速率。     

血红蛋白在海藻酸钠膜中的电化学和类酶活性研究

用海藻酸钠(SA)将血红蛋白(Hb)固定到热裂解石墨电极表面,制备了Hb-SA膜修饰电极。包埋在SA膜中的Hb在磷酸盐缓冲溶液和乙醇混合溶液中与电极直接传递电子,得到一对对称的血红蛋白辅基血红素Fe(Ⅲ)/Fe(Ⅱ)电对的可逆氧化还原峰。其式电势随缓冲溶液pH值增加而负移,且呈线性关系,这说明血红蛋白的电子传递过程伴随有质子的转移。并考察了Hb-SA膜修饰电极在缓冲溶液和乙醇混合溶液中对氧气、双氧水、一氧化氮和六氯乙烷的电催化性质。     

阿魏酸修饰的血红蛋白自氧化的研究(加急)

为研究FA-Hb的自氧化,以阿魏酸酯（FA-NBA）与血红蛋白（Hb）为原料,制备了阿魏酸血红蛋白（FA-Hb）。通过紫外光谱扫描、血氧分析仪等检测手段,分析了修饰前后血红蛋白的特征吸收峰的变化情况和氧饱和曲线的差别;研究了有无叠氮钠、黄嘌呤-黄嘌呤氧化酶介导下,血红蛋白和阿魏酸修饰的血红蛋白的自氧化速率的改变。实验结果表明,阿魏酸修饰血红蛋白,保持了血红蛋白的功能结构,仍具备血红蛋白的载氧活性,有效降低了自氧化速率。     

氮氧自由基定点修饰牛血红蛋白的制备和抗自氧化性能

以4-(N-马来酰亚胺基)-2,2,6,6-四甲基哌啶-1-氮氧自由基(Mal-Tempol)和牛血红蛋白(Hb)为原料,制备了氮氧自由基定点修饰在巯基上的血红蛋白(Mal-Tempol-Hb)。利用分光光度法测定了氮氧自由基的修饰度,通过紫外光谱考察了有无叠氮化钠(NaN_3)介导下,Hb和Mal-Tempol-Hb中高铁血红蛋白(Met Hb)含量随时间变化的过程。结果表明,无NaN_3介导下,180 min时,Mal-Tempol-Hb中Met Hb的摩尔分数由修饰前的44%下降为修饰后的28%;在NaN_3介导下,180 min时,Mal-Tempol-Hb中Met Hb的摩尔分数从修饰前的50%下降为修饰后的32%。氮氧自由基的修饰有效地降低了血红蛋白的自氧化速率。     

陶瓷膜微滤-聚合物强化超滤处理高浓度含镍废水

实验研究了0.5 mm孔径陶瓷膜对高浓度含镍废水的微滤行为,以聚丙烯酸钠(PAAS)强化超滤技术深度处理陶瓷膜渗透液,考察了PAAS与金属质量比(rp/m)和pH值对恒容超滤膜通量(J)和镍截留系数(RNi)的影响,研究了超滤浓缩、解络合、洗涤及PAAS循环使用过程. 结果表明,pH=9时,陶瓷膜浓缩时J先快速降低、缓慢下降后再较快降低,RNi接近1,当体积浓缩因子从1增大到10时,截留液镍浓度(Cr)从5562.71 mg/L浓缩至55507.76 mg/L,渗透液镍浓度(Cp)为13.26 mg/L. PAAS强化恒容超滤时,J不随rp/m变化,随pH值增大而增大,RNi随rp/m或pH值增大而增大;超滤浓缩时,控制pH=9和rp/m=9,RNi接近1,Cr呈线性递增,Cp"0.05 mg/L;在pH=3条件下对超滤浓缩液解络合,解离平衡时间为9 min,解络合率为81.9%;以pH=3的盐酸溶液洗涤解络合液,镍洗脱率为98.8%. 再生PAAS络合性能良好,可循环使用.     

颗粒粒度对Cu基载氧体反应性能的影响

基于化学链燃烧新流程,采用固定床反应器研究了Cu基载氧体粒度、作为活性组分的Cu粉粒度及作为惰性载体的g-Al2O3粒度对载氧体反应性能的影响. 结果表明,载氧体的还原率及氧化率随Cu粉粒度减小而增大,Cu粉平均粒度小于63 mm时,载氧体还原率和氧化率达95%和93%以上. 载氧体还原过程中有少量CH4生成,生成量随Cu粉粒度减小而减少,Cu粉平均粒度小于32 mm时低于1%;当载氧体粒度接近g-Al2O3粒度时,载氧体的性能明显降低,只有g-Al2O3粒度明显小于载氧体粒度时,g-Al2O3才能起到分散活性组分、抑制活性组分烧结的作用.     

膨胀床吸附高效纯化牛血红蛋白

探索了将膨胀床吸附层析应用于动物血液蛋白质的提取,尝试了直接从牛血红细胞破碎液中纯化血红蛋白.对该过程采用的吸附介质及pH值、离子强度、温度、洗脱条件等因素对高铁血红蛋白生成、产品纯度以及回收率等的影响进行探索.选择了最佳吸附介质Streamline SP,确定了优化的吸附条件为：吸附过程选用离子强度为10mmol•L^-1的磷酸盐缓冲系统,进料线流速3.3cm•min^-1,进料pH值6.6,改变缓冲液pH值为7.2进行洗脱,操作温度4℃.该技术省去了离心去除碎片步骤,有效地控制了纯化过程中无载氧能力的高铁血红蛋白的生成,动态吸附容量达到70～75mg•ml^-1,只经一步操作产品纯度达电泳纯,高铁血红蛋白含量仅为5.4％.与现有的膜过滤-层析纯化方法相比,膨胀床吸附法操作时间短,提取收率高,产品活性损失小,是一种简捷、高效、适合工业放大的天然血红蛋白制备方法.     

乙二醇二缩水甘油醚交联血红蛋白的反应优化

将乙二醇二缩水甘油醚(EGDE)作为血红蛋白(Hb)的交联剂制备红细胞代用品.采用SDS-PAGE电泳、高效液相色谱和多角度激光散射检测器联用鉴定交联产物.实验表明,在pH 8.7,血红蛋白浓度为20 mg·mL-1,修饰剂EGDE与血红蛋白的摩尔比达100:1时反应4 h,交联产物中90%为分子内交联的血红蛋白.在pH 7.5、血红蛋白浓度为200mg·mL-1,修饰剂EGDE与血红蛋白的摩尔比为25:1时反应12 h,交联产物主要为寡聚血红蛋白,分子量范围在145-375 kD之间;其中二聚体血红蛋白占总蛋白量的74.7%,三聚体血红蛋白18.9%,四到六聚血红蛋白6.4%,几乎无高聚物生成.采用血氧分析仪对交联产物进行活性鉴定,制备的分子内交联和寡聚血红蛋白均具有携氧能力,小鼠的生物学实验表明产品无异常毒性,具备了作为红细胞代用品的可能性.     

Determination of Methemoglobin in Hemoglobin Submicron Particles Using NMR Relaxometry

Methemoglobin (MetHb) is a hemoglobin (Hb) derivative with the heme iron in ferric state (Fe³⁺), unable to deliver oxygen. Quantification of methemoglobin is a very important diagnostic parameter in hypoxia. Recently, novel hemoglobin microparticles (Hb-MP) with a narrow size distribution around 700 nm, consisting of cross-linked Hb were proposed as artificial oxygen carriers. The cross-linking of Hb by glutaraldehyde (GA) generates a certain amount of MetHb. Due to the strong light scattering, quantitative determination of MetHb in Hb-MP suspensions by common spectrophotometry is not possible. Here, we demonstrate that ¹H₂O NMR relaxometry is a perfect tool for direct measurement of total Hb and MetHb concentrations in Hb-MP samples. The longitudinal relaxation rate 1/T₁ shows a linear increase with increasing MetHb concentration, whereas the transverse relaxation rate 1/T₂ linearly increases with the total Hb concentration. In both linear regressions the determination coefficient (R²) is higher than 0.99. The method does not require time-consuming pretreatment or digestion of the particles and is not impaired by light scattering. Therefore, it can be established as the method of choice for the quality control of Hb-MP and similar hemoglobin-based oxygen carriers in the future.     

Development of a biosensor for glycated hemoglobin

The development of an electrochemical piezoelectric sensor for the detection of glycated hemoglobin is presented. The total hemoglobin (Hb) content is monitored with a mass-sensitive quartz crystal modified with surfactants, and the glycated fraction of the immobilized Hb is determined by subsequent voltammetric measurement of the coupled ferroceneboronic acid.Different modifications of the sensor were tested for their hemoglobin binding ability. Deoxycholate (DOCA) was found to be the most suitable among the examined modifiers. Piezoelectric quartz crystals with gold electrodes were modified with DOCA by covalent binding to a pre-formatted 4-aminothiophenol monolayer. The properties of the Hb binding to DOCA and the pH effect on this interaction were studied.In the proposed assay for glycated hemoglobin at first an Hb sample is incubated with ferroceneboronic acid (FcBA), which binds to the fructosyl residue of the glycated Hb. Then this preincubated Hb sample is allowed to interact with the DOCA-modified piezoelectric quartz crystal. The binding is monitored by quartz crystal nanobalance (QCN). The amount of FcBA present on the sensor surface is determined by square wave voltammetry. The binding of FcBA results in well-defined peaks with an E⁰′ of +200 mV versus Ag/AgCl (1 M KCl). The peak height depends on the degree of glycated Hb in the sample ranging from 0% to 20% of total Hb. The regeneration of the sensing surface is achieved by pepsin digestion of the deposited Hb. Thus the sensor can be re-used more than 30 times.     

Iron and vitamin A deficiencies and prevalence of anemia in boys and girls between 6 to 24 months of age in Chaco,Argentina

To determine the prevalence of anemia, and iron (ID) and vitamin A deficiencies aiming at their prevention, 414 children between 6 and 24 months of age, were randomly selected from the whole province of Chaco. A sociodemographic survey was implemented, and hemoglobin (Hb), plasma ferritin and retinol were measured. Anemia prevalence (Hb < 110 g/L) was 66.4%, without differences between age groups, and included 18% with Hb < 90 g/L. These cases were significantly less in children 6-8 month of age (5.1%) than in the others (approximately equal to 20%) (P: 0.007). Mean Hb was also higher in 6-8 months old children and was associated with lower prevalence of ID (ferritin < 12 micrograms/L) (p < 0.000) but not with age (p = 0.8865). ID already present, however, in 36.6% of children in this age group, reached a prevalence of 72.9% in children older than 18 months. Anemia prevalence was significantly higher in males, in children whose birth weight was < 3000 g, in those who had never taken iron supplements and among the poor, both structural and by income. Retinol values < 20 micrograms/dl occurred only in 5.1% of children. Iron nutrition prior to, during pregnancy and in children during the first 2 years of life must be improved by joining strategies based on community empowerment aimed at improving dietary iron, assuring effective preventive supplementation and promoting the opportune umbilical cord ligation.     

大枣多糖与山楂乙醇提取物协同抗氧化研究

用清除2,2′-连氨-(3-乙基苯并噻唑啉-6-磺酸)二氨盐(ABTS)自由基法,研究了大枣多糖(JDP)、山楂乙醇提取物、JDP与山楂乙醇提取物组合的复合物的协同抗氧化作用。综合运用加和法和直接比较法分析了复合物的协同作用,用响应曲面法分析讨论了单一组分及协同作用对复合物的抗氧化活性的影响。清除ABTS自由基的实验结果表明:清除氧自由基能力的大小顺序为:SC>SD>SB>DW>SE>SA;其中SC(IC50=2.51 mg/L)的抗氧化活性最强,JDP与SC和JDP与SE分别具有协同抗氧化活性。     

Limitations of the method using peroxidase activity of hemoglobin for detecting lipid hydroperoxides

The method using peroxidase activity of hemoglobin (Hb) for the determination of lipid peroxides was examined by using pure methyl linoleate hydroperoxides, trilinoleoylglycerol hydroperoxides and egg yolk phosphatidylcholine hydroperoxides as substrates and tetramethyl benzidine as electron donor for the peroxidase reaction of Hb. The reactivities of these substrates were quite varied. Furthermore, some electron donors were tested for peroxidase activity of Hb, but none showed a complete reduction of methyl linoleate hydroperoxides. From these results, it seems the Hb method needs to be carefully applied to biological materials that contain mixtures of different types of lipid classes.     

Electrochemical sensors for hemoglobin and myoglobin detection based on methylene blue-multiwalled carbon nanotubes nanohybrid-modified glassy carbon electrode

An effective electrochemical sensors for hemoglobin (Hb) and myoglobin (Mb) detection was firstly developed using a simple procedure of self-assembled methylene blue-multiwalled carbon nanotubes (MB-MWNTs) nanohybrid modified on glassy carbon electrode without using any enzymes immobilization. The direct electrochemical and electrocatalytic behaviors of the modified electrode were studied using cyclic voltammetry (CV) and flow injection analysis (FIA) with amperometry. The performance of the sensor was investigated and optimized and the system was evaluated by monitoring Hb and Mb concentrations. The developed MB-MWNTs nanohybrid modified electrode showed excellent electrocatalytic activity for reduction of Hb and Mb with good stability, sensitivity and reproducibility (RSD = 3.05% and 4.5% for 50 successive injections of Hb and Mb, respectively). Under optimal conditions, the catalytic currents are linearly proportional to the concentrations of Hb and Mb in the wide range from 5 nM to 2 μM and 0.1 to 3 μM, and the corresponding detection limits are 1.5 nM and 20 nM (S/N = 3), respectively. This approach provides improved detection limit over other previous works and may provide a novel and efficient platform for the fabrication of sensors for other heme proteins.     

Direct electrochemistry and electrocatalysis of hemoglobin with carbon nanotube-ionic liquid-chitosan composite materials modified carbon ionic liquid electrode

A novel composite biomaterial was prepared by combining chitosan, multi-walled carbon nanotubes (MWCNTs), hemoglobin (Hb) and ionic liquid (IL) 1-butyl-3-methyl-imidazolium bromide together, which was further modified on the surface of a carbon ionic liquid electrode (CILE) with another ionic liquid 1-ethyl-3-methylimidazolium ethylsulphate as the binder. Ultraviolet-visible and Fourier transform infrared spectroscopic results indicated that Hb molecules in the composite film retained the native structure. Cyclic voltammetric results showed that a pair of well-defined redox peaks appeared in 0.1 mol/L phosphate buffer solution, indicating that the direct electron transfer of Hb in the composite film with the underlying electrode was realized. The results were attributed to the synergistic effect of MWCNTs and IL in the composite film, which promoted the electron transfer rate of Hb. The composite material modified electrode showed excellent electrocatalytic ability towards the reduction of different substrates such as trichloroacetic acid and NaNO₂ with good stability and reproducibility.     

Oxygen binding and other physical properties of human hemoglobin made in yeast

Wagenbach et al. (1991, BioTechnology, 9, 57–61) haverecently developed a system for producing soluble recombinanttetrameric hemoglobin in yeast: hemoglobin begins to appear4– 5 h after induction with galactose,- and ß-globinchains fold in vivo and endogeneously produced heme is incorporatedinto hemoglobin tetramers. We have further characterized theoxygen-binding properties, as well as the tetramer stability,of recombinant human Hb A made in yeast. After purificationby ion-exchange chromatography, a single band at the same positionas normal human Hb A was obtained using cellulose acetate electrophoresis.Although the oxy and deoxy forms of purified recombinant HbA made in yeast were spectrophotometrically identical to nativehuman Hb A, the oxygen-binding curve was shifted slightly leftof that for native human Hb A. Further purification of recombinanthemoglobin by FPLC revealed two fractions: one (fraction B)with low cooperativity and high oxygen affinity, and the other(fraction A) with almost identical cooperativity and oxygenaffinity compared with native human Hb A. The Bohr effect offraction A was also identical to native human Hb A. Hemoglobinin fraction B with lowered cooperativity precipitated -1.5 timesfaster than normal human Hb A during mechanical agitation, whilehemoglobin in fraction A with normal cooperativity precipitatedwith kinetics identical to native human Hb A. These resultssuggest that some of the recombinant molecules made in yeastfold improperly, and that these molecules may exhibit decreasedcooperativity for oxygen binding and decreased stability. Thissystem should now allow the evaluation of the folding differenceswhich promote hemoglobin function, and should also provide anefficient system which will facilitate the production of varioushemoglobin mutants for studying biochemical and biophysicalproperties of hemoglobin.     

Direct electrochemistry and electrocatalysis of hemoglobin immobilized in poly(ethylene glycol) grafted multi-walled carbon nanotubes

The novel poly(ethylene glycol) (PEG) grafted multi-walled carbon nanotubes (PEG-g-MWCNTs) were synthesized by the covalent functionalization of MWCNTs with hydroxyl-terminated PEG chains, exhibited excellent hydrophilicity and biocompatibility. The PEG-g-MWCNTs were characterized by Fourier transform infrared spectra, transmission electron microscopy, and thermogravimetric analysis, which verified that PEG chains were grafted onto the surface of the MWCNTs. The PEG-g-MWCNTs were then used as substrates for the immobilization of hemoglobin (Hb) and their bioelectrochemical behaviors were studied. Electrochemical impedance spectroscopy was used to confirm the adsorption of Hb onto the surface of PEG-g-MWCNTs. The Hb immobilized in PEG-g-MWCNTs retained its near-native conformations as characterized by the UV-vis spectroscopy. The cyclic voltammetry results of Hb/PEG-g-MWCNT modified electrode showed a pair of well-defined and quasi reversible redox peaks centered at approximate −0.34 V (vs. saturated calomel electrode), which was the characteristic peaks of Hb Fe(III)/Fe(II), in pH 7.0 phosphate buffer solution. Hb immobilized onto the surface of PEG-g-MWCNTs demonstrated good bioelectrocatalytic activities for the reduction of nitrite.     

Protective factors for iron deficiency anemia: prospective study in low-income infants

This study was performed to assess both risk and protective factors associated with iron deficiency anemia in 130 infants with age below 24 months, with low socio-economic status and followed since their births by a primary health care program in the city of S?o Paulo, Brazil. Growth, morbidity and dietary factors were analysed as well as hemoglobin levels (Hb) at regular intervals (6, 9, 12, 18 and 24 months). The portable Hemocue photometer was employed to measure hemoglobin levels and anemia was considered when Hb values were below 11 g/dL. Simple logistic regression was used with socio-economic variables, age group, nutritional status, morbidity, breastfeeding and food intake. Risk factors for anemia in the studied group were intake of cow's milk at 4 months and who has 3 or more old brothers (OR approximately = 2). The protective factors for anemia were age between 18 and 24 months and intake of infant formula, vitamin C, meat and beans at 6 months (OR approximately = 0.5). The prevention of iron deficiency anemia includes encouraging healthy feeding practices starting at 6 months of life.