Electron Spin Resonance Evidence of the Generation of Superoxide Anion, Hydroxyl Radical and Singlet Oxygen during the Photohemolysis of Human Erythrocytes with Bacteriochlorin a

Abstract— Photodynamic therapy with bacteriochlorin a (BCA) as sensitizer induces damage to red blood cells in vivo. To assess the extent of the contributuion of reactive oxygen species (ROS) and to determine a possible reaction mechanism, competition experiments with assorted ROS quenching or/and enhancing agents were performed in human erythrocytes as model system and in phosphate buffer. In the erythrocyte experiments, a 2% suspension was incubated with BCA for 1 h, washed with phosphate-buffered saline, resuspended and subsequently illuminated with a diode laser using a fluence rate of 2.65 mW/cm₂. Potassium leakage and hemolysis were light and BCA dose dependent. Adding tryptophan (3.3 mM), azide (1 mM) or histidine (10 mM) to the erythrocyte suspension before illumination delayed the onset of K-leakage and hemolysis suggesting a type II mechanism. The D₂O did not affect K-leakage nor photohemolysis. Adding mannitol (13.3 mM) or glycerol (300 nM) also caused a delay in the onset of K-leakage and hemolysis, suggesting the involvement of radicals. In phosphate buffer experiments, it was shown using electron spin resonance (ESR) associated with spin-trapping techniques that BCA is able to generate 0₂～* and OH* radicals without production of aqueous electron. Visible or UV irradiation of the dye in the presence of the spin trap 5,5-dimethyl-1-pyrroline-iV-oxide (DMPO) gave an ESR spectrum characteristic of the DMPO-hydroxyl radical spin adduct DMPO-OH. Addition of ethanol or sodium formate produced supplementary hyperfine splittings due to the respective CH₃CHOH _* and CO_2-1 radical adducts, indicating the presence of free OH_*. Production of DMPO-OH was partly inhibited by superoxide dismutase (SOD), catalase and desferoxamine, suggesting that the iron-catalyzed decomposition of H₂O₂ was partly involved in the formation of one part of the observed OH _*. The complementary inhibition of DMPO-OH production by azide and 9,10-anthracenedipropionic acid (ADPA) was consistent with ₁O₂ production by BCA followed by reaction of ₁O₂ with DMPO and decay of the intermediate complex to form DMPO-OH and free OH_*. All our results seem to indicate that BCA is a 50%/50% type 1/type 2 sensitizer in buffered aqueous solutions and confirmed that the dye-induced hemolysis of erythrocytes was well caused by a mixed type 1/type 2 mechanism.     

Effect of Membrane Potential on the Binding of Merocyanine 540 to Human Erythrocytes

Illumination of erythrocytes in the presence of merocyanine 540 (MC540) resulted in changed binding characteristics of MC540, i.e. a red shift in the emission maximum of bound dye with an increase in the relative fluorescence quantum yield. Aluminum phthalocyanine tetrasulfonate-mediated photodynamic treatment, before addition of MC540, resulted in a comparable change in the MC540-binding characteristics with, in addition, an increase in the concentration of MC540 in the membrane. Both photodynamic treatments induce depolarization of the red cell membrane, with a dose dependency comparable to that of changed MC540 binding. Also depolarization, induced by incubation of the cells with A23187 in the presence of Ca₂+ in high [K+] buffer, resulted in similar changes in the MC540 binding characteristics. These results indicate a relation between photodynamically induced membrane depolarization and changed MC540-binding characteristics. Hyperpolarization induced by incubation with A23187 in low [K+] buffer resulted in decreased binding of MC540. In accordance, the MC540-mediated photodamage to red cells decreased upon hyperpolarization of the cells. The results indicate that the binding of MC540 to erythrocytes is strongly dependent on the membrane potential and that hyperpolarization of the membrane could be a possible protection mechanism for erythrocytes against MC540-mediated photodynamic damage.     

Effects of the Oxovanadium Compounds on Human Erythrocytes

In recent years, vanadium compounds have attracted the interest of many researchers due to their potential use in diabetes treatment. In order to improve their therapeutic effect, quite a lot of vanadium compounds were synthesized and screened. The structure-activity relationship has been explored. However, the actual therapeutic effect is not only dependent on the pharmacological action, but also on whether an effective level could be established in the target site. In the present work, the bioavailability and the toxicity of the different oxovanadium confounds were studied using human erythrocytes as a model system.     

Cesium's Affects on Morphological Changes of Human Erythrocytes

Cesium could play a toxic role in several pathological processes. Atomic force microscopy (AFM) was used to study morphological changes of human erythrocytes after incubating with different concentrations of CsCl, and the Raman spectra were used to study the effects of CsCl on the chemistry components of erythrocyte membrane. The AFM images showed that the "domain structures" that appeared after incubation with higher concentration of CsCl (150 mmol·L^?1), are featured by the particles aggregated to form ranges and the separations among them enlarged to gorges, and this change may increase the permeability of cell membranes. The Raman results showed that the polar part of membrane phospholipid become more order and with the increasing of the concentration of CsCl, the longitudinal order of nonpolar parts first decreased and then increased. It is concluded that the aggregation of membrane proteins and the order changes of the phospholipid cause a change in the distribution and conformation of the phospholipid membrane. And the effects of CsCl on the erythrocyte membrane are mainly dependent on its concentration.     

Effect of Magnetite Particles on Photoinduced and Nonphotoinduced Free Radical Processes in Human Erythrocytes

Magnetite (Fe₃0₄) encapsulated in polystyrene microspheres dramatically decreased the time for 50% hemolysis (t_1/2) of human erythrocytes irradiated (λ300 nm) in the presence of ketoprofen (0.1 mM). The magnetic microspheres were present at a very low concentration (0.002%) such that on average there was only one particle per four erythrocytes. No such effect was seen when nonmagnetic microspheres were employed or when the equivalent concentration of soluble iron (FeCl₃) was present. A decrease in t_1/2 was also observed when the magnetic microspheres were added after UVA/ketoprofen treatment or when they were present during hemolysis initiated by thermolysis of 2,2′-azobis(2-amidinopro-pane). These findings may be attributed to an increase in the membrane concentration of lipid radicals as a result of a magnetic field-induced increase in radicals escaping from triplet radical pairs.     

两种含氮类大豆苷元衍生物对人体红细胞的抗溶血作用

合成了2种含氮类大豆苷元衍生物,4,7-二（（甘氨酸钠）羰基）甲氧基异黄酮（L1）和4'',7-二（肼羰基）甲氧基异黄酮（L2）并用元素分析、红外光谱和核磁共振氢谱对其进行了表征。研究了大豆苷元衍生物的抗氧化性能,评价了其对自由基、超氧阴离子自由基的吸附能力,以及对人体血红细胞抗氧化损伤的保护作用。实验结果表明,大豆苷元衍生物在生理pH条件下的抗氧化活性优于维生素C,特别是在清除羟自由基和抑制人血红细胞溶血方面,大豆苷元衍生物抗氧化能力表现更为突出。大豆苷元衍生物的羟自由基清除活性IC₅₀值是维生素C的104倍。     

Photohemolysis sensitized by the furocoumarin imperatorin and its oxyfunctionalized derivatives

The dark and photosensitized (366 nm) hemolytic effects of imperatorin and its photooxidation products, the hydroperoxides I and II as well as the corresponding alcohol of the hydroperoxide I (imperatorin alcohol), were studied on human erythrocytes. Imperatorin was shown to photosensitize hemolysis, its fluence (D) dependence of the rate of photohemolysis (V) followed the equation V = V0 + aD2 + bD1/2, in which V0 is the dark hemolysis rate and a and b are constants. At fluences below 200 kJ/m2, the main hemolytic contribution derives from the bD1/2 component, which is due to the in situ formation of the imperatorin hydroperoxides, while at fluences higher than 200 kJ/m2, the main contribution corresponds to the aD2 component due to the two-photon damage of cell membranes. Hydroperoxides I and II induce oxyhemoglobin cross-linking, as well as its conversion to methemoglobin and hemichrome. These reactions involve hydroxyl and alkoxy radicals, as the hemolysis and oxyhemoglobin conversion could be inhibited by t-butanol and butylated hydrotoluene. For comparison, the dark hemolytic effect of the imperatorin alcohol was approximately 10-fold less than of the hydroperoxides.     

重稀碱金属Cs~+跨人红细胞膜行为的研究(英文)

采用原子吸收光谱法检测体外人红细胞摄取Cs+的含量,系统讨论了胞外Cs+浓度,温育时间、温育温度、介质pH值对人红细胞摄取Cs+过程的影响。选用不同离子通道或离子载体的特异性抑制剂进一步探讨Cs+的跨膜途径和机理。结果显示,各实验参数对人红细胞摄取Cs+均有一定的促进作用。Cs+主要借助Na+/K+-泵的主动运输方式跨膜;少量的Cs+能"漏入"细胞,微量的Cs+可以模拟Na+/Li+-反向协同运输的方式跨膜;在允许HCO3-存在的pH环境下,少量Cs+以Cl-/CsCO3-交换的形式通过膜上带3蛋白进入人红细胞;Ca2+通道对Cs+没有通透作用。     

Rb+跨人红细胞膜的研究

The erythrocyte hemolysis was examined to assure Rb^＋ to have a weak toxicity toward human body. The way of Rb^＋ transporting into human erythrocytes was determined, and the factors to affect this transport process were evaluated. The effects of extracellular concentration of Rb^＋, incubation temperature, incubation time, medium pH, and specific inhibitors were investigated via flame atomic absorption spectrometry. The results indicated that the membrane transport of Rb^＋ through human erythrocytes was controlled mainly by both active transport and simple diffusion. Every mentioned factor took a positive effect on the Rb^＋ uptake by human erythrocytes, however neither DIDS nor nefidpine could inhibit the uptake of Rb^＋.     

Purification of Acetylcholinesterase by 9-Amino-1,2,3,4-tetrahydroacridine from Human Erythrocytes

The acetylcholinesterase enzyme was purified from human erythrocyte membranes using a simple and effective method in a single step. Tacrine (9-amino-1,2,3,4-tetrahydroacridine) is a well-known drug for the treatment of Alzheimer's disease, which inhibits cholinesterase. We have developed a tacrine ligand affinity resin that is easy to synthesize, inexpensive and selective for acetylcholinesterase. The affinity resin was synthesized by coupling tacrine as the ligand and l-tyrosine as the spacer arm to CNBr-activated Sepharose 4B. Acetylcholinesterase was purified with a yield of 23.5 %, a specific activity of 9.22 EU/mg proteins and 658-fold purification using the affinity resin in a single step. During purification, the enzyme activity was measured using acetylthiocholine iodide as a substrate and 5,5′-dithiobis-(2-nitrobenzoicacid) as the chromogenic agent. The molecular weight of the enzyme was determined as about 70 kDa monomer upon disulphide reduction by sodium dodecyl sulphate polyacrylamide gel electrophoresis. K _m, V _max, optimum pH and optimum temperature for acetylcholinesterase were found by means of graphics for acetylthiocholine iodide as the substrate. The optimum pH and optimum temperature of the acetylcholinesterase were determined to be 7.4 and 25–35 °C. The Michaelis–Menten constant (K _m) for the hydrolysis of acetylthiocholine iodide was found to be 0.25 mM, and the V _max was 0.090 μmol/mL/min. Maximum binding was achieved at 2 °C with pH 7.4 and an ionic strength of approximately 0.1 M. The capacity for the optimum condition was 0.07 mg protein/g gel for acetylcholinesterase.     

Photohemolysis Sensitized by the Furocoumarin Derivative Alloimperatorin and its Hydroperoxide Photooxidation Product

The dark and photosensitized effects of alloimperatorin methyl ether 1 (hereafter simply alloimperatorin) and its photooxygenation product alloimperatorin hydroperoxide 2 were investigated on human erythrocytes. The results reveal that the furocoumarin 1 photosensitizes efficiently the hemolysis of erythrocytes. The rate of photohemolysis increases on raising the temperature of the postirradiated incubation from 4°C to 37°C. Thermal activation of the photohemolysis and inhibition by 2,6‐di‐tert‐butyl‐p‐cresol (BHT) suggest that the furocoumarin 1 photosensitizes lipid peroxidation, increasing permeability in the erythrocyte membrane. The hydroperoxide 2 induces dark and photosensitized hemolysis more efficiently than the furocoumarin 1. The rate of hemolysis induced by 2 increases with the incubation temperature and decreases in the presence of tert‐butanol and BHT. The hydroperoxide 2 photosensitizes the formation of lipid peroxidation products as shown by the reaction with thiobarbituric acid. This process is diminished by BHT. Our data imply that the photohemolysis sensitized by the furocoumarin 1 is caused by the in situ‐formed photooxygenation product 2. Such hydroperoxides are potent hemolytic agents in the dark and especially on photosensitization.     

高效液相色谱法测定人体红细胞中儿茶酚氧位甲基转移酶的活性

 采用高效液相色谱紫外检测法测定人体红细胞中儿茶酚氧位甲基转移酶 (COMT)的活性。以 3,4 二羟基苯甲酸 (DBA)作为酶反应底物 ,S 腺苷甲硫氨酸 (SAM)作为甲基供体 ,在镁离子的存在下 ,将SAM上的甲基转移到DBA 3位的氧上。色谱法测定反应产物 4 羟基 3 甲氧基苯甲酸 (4 OH 3 MBA)的生成量。人体红细胞中COMT活性的线性范围在 1U/mL～ 6 0U/mL ,最低检测限为 0 5U/mL(S/N≥ 5 ) ,方法的精密度良好 (平均RSD <10 % )。     

电化学方法研究环糊精与酮洛芬的包结行为

以电化学方法研究了各种环糊精及其衍生物对酮洛芬电化学行为的影响，测定了酮洛芬与β环糊精，羟丙基β－环糊精和甲基化－β－环糊精的包结常数及其温度对包结作用的影响，并由各包结物的热力学参数解释包结现象。     

旋转磁场对质子交换膜燃料电池工作性能的影响

外加磁场可以提高PEM（Proton Exchange Membrane,质子交换膜）燃料电池的工作性能,本文通过在PEM燃料电池表面分别附加正方形梯度磁场、同极性组合圆柱形磁场以及异极性组合圆柱形磁场,分析旋转磁场、静态磁场以及未加磁场3种磁场环境下燃料电池的输出功率密度变化. 研究发现,外部磁场的分布规律不同,磁场的变动对燃料电池的影响也不相同,尤其是异极性组合磁场,旋转的磁场使燃料电池的最大功率密度提高了21.27%,明显高于加载静态磁场时提高的11.70%. 旋转磁场产生的效果与磁场旋转速度有关,提高转速有利于增强磁场对燃料电池工作性能的影响,当转速为30 r·min^-1时影响最大,随着转速进一步提高,影响效果逐渐变差。     

The Kabachnik–Fields Reaction Accelerated in External Magnetic Field

The significant acceleration in the Kabachnik–Fields reaction in an external magnetic field has been observed. The phenomenon is explained by the proper orientation of substrate molecules forced by external magnetic field. Diamagnetic dipoles are repulsed to the center by both electromagnet poles, being simultaneously arranged. This phenomenon may then promote the acceleration of the reaction.     

The Application of UV-Radioactivity High Performance Liquid Chromatography to the Study of Hypoxanthine Transport in Human Erythrocytes

Abstract

A procedure is presented for the simultaneous measurement of concentrations of labeled and non labeled hypoxanthine by HPLC in order to study hypoxanthine transport in erythrocytes. A radioactivity detector connected on-line to the high performance liquid chromatograph in series with a UV detector provides on-line quantitative monitoring of hypoxanthine in erythrocytes or incubation medium. The procedure provides a rapid, sensitive and convenient means for the study of hypoxanthine transport.     

外加磁场条件下镍纳米材料的形貌可控制备

在常压下,采用外加磁场辅助的液相还原法制备得到了直径约为250 nm镍纳米晶纤维。磁性能测试结果表明镍纤维的饱和磁化强度为8.94 emu·g^-1,矫顽力为133.8 Oe,说明得到的镍纤维具有铁磁性能。此外,还研究了溶剂种类对产物微观结构和形貌的影响。结果表明,产物的形貌受溶剂种类的影响十分明显,只有分子结构中含有2个端羟基的醇类溶剂才可以用来制备形貌均一的超细镍纤维。     

人红细胞摄入Cd~(2 )离子的动力学和机理

不同Cd2 离子浓度引起的人红细胞相对溶血率及临界溶血浓度CH0 已经测定 ,CH0 =2 .5× 10 - 4mol·L- 1。Cd2 跨膜进入人红细胞遵从一级动力学过程 ,其一级速率常数为 1.2× 10 - 2 h- 1。阴离子通道抑制剂DIDS及K ,Na ATPase抑制剂均可部分抑制人红细胞摄入Cd2 ,Cl- 浓度和介质pH也影响Cd2 的细胞摄入 ,细胞摄入镉量随胞外Cd2 浓度增加线性增加。这些实验结果证明 ,人红细胞摄入Cd2 是涉及简单被动扩散、阴离子通道转运及主动传送的复杂过程     

The Influence of Combined Magnetic and Vibrowave Fields on the Thermal Conductivity and Strength of Filled Polymer Adhesives

The results of the experimental study of the effect of the combined effect of a constant magnetic field and low-frequency mechanical oscillations on the coefficient of thermal conductivity of adhesive interlayers and the shear strength of adhesive joints are presented. It is shown that the action of the combined field leads to the modification of polymer adhesives filled with nickel and iron powders, which makes it possible to make adhesive joints with higher thermal conductivity and strength properties.     

酮洛芬对映体的双相识别手性萃取分离

研究了酮洛芬在溶有酒石酸酯的有机相和β-环糊精衍生物水相萃取体系中的分配行为;考察了有机溶剂、酒石酸酯、环糊精衍生物的种类、萃取剂浓度和pH等因素对分离效果的影响。结果表明,β-环糊精衍生物优先识别S-对映体而不是R-对映体,但L-酒石酸酯的识别能力刚好相反;1,2-二氯乙烷作为有机溶剂,三甲基-β-环糊精和L-酒石酸异丁酯做手性萃取剂是最好的选择;萃取剂的浓度和pH对分离效果有明显影响;当三甲基-β-环糊精和L-酒石酸异丁酯的浓度分别为0.1mol/L和0.2mol/L、水相pH=2.5时,分离效果最好。