利福平及其蛋白偶联物致敏兔和利福平特异IgG的检测

用利福平及两种利福平－蛋白偶联物：利福平－牛血清白蛋白及利福平－－兔血清白蛋白免疫兔，用ELISA方法对兔抗血清中利福平特异IgG进行检测。利福平－牛血清白蛋白及利福平－兔血清白蛋白的偶联方式及偶联比例不同。利福平特异IgG的效价分别为：利福平免疫组1：10－1：100，利福平－兔血清白蛋白免疫组1：100－1：10000；利福平－牛血清白蛋白免疫组超过1：1000。抑制实验证明了抗血清的利福平特异性。这些利福平致敏动物模型是首次用利福平或利福平－蛋白偶联物致敏的动物模型。     

紫外光谱研究中药大黄有效成分与牛血清白蛋白的相互作用

目的　用紫外光谱研究大黄有效成分 :大黄素、大黄酸和大黄酚与牛血清白蛋白的相互作用 ,了解大黄有效成分的生物结合活性。方法　采用紫外光谱法 ,通过测量加入牛血清白蛋白后药物吸光度值的变化 ,以Scatchard方程作图求解。结果　大黄三种主要有效成分 :大黄素、大黄酸和大黄酚与牛血清白蛋白相互作用的结合常数分别为K =1 .4 7× 1 0 5,K =5 .0 0× 1 0 5,K =1 .1 8× 1 0 4。其中大黄素与牛血清白蛋白的结合常数与文献值较一致 ,大黄酸、大黄酚与牛血清白蛋白的结合常数未见报道。结论　从结合机制上对测定结果进行了探讨 ,大黄类有效成分与牛血清白蛋白的结合能力随其所含极性基团的增多而增强。表明该方法研究药物 蛋白相互作用简便、快速、可行。     

利福平及其蛋白偶联物致敏兔和利福平特异IgG检测(英文)

用利福平及两种利福平－蛋白偶联物：利福平－牛血清白蛋白及利福平－兔血清白蛋白免疫兔，用ＥＬＩＳＡ方法对兔抗血清中利福平特异ＩｇＧ进行检测。利福平－牛血清白蛋白及利福平－兔血清白蛋白的偶联方式及偶联比例不同。利福平特异ＩｇＧ的效价分别为：利福平免疫组ｌ：ｌ０～１：ｌ００；利福平－兔血清白蛋白免疫组１：１０～ｌ：ｌ０００；利福平－牛血清白蛋白免疫组超过１：１０００。抑制实验证明了抗血清的利福平特异性。这些利福平致敏动物模型是首次用利福平或利福平－蛋白偶联物致敏的动物模型。     

用于卡介菌多糖核酸制剂效力实验的小鼠模型的建立

目的  建立用于卡介菌多糖核酸制剂效力实验的小鼠模型。方法  选用猪血清、牛血清白蛋白、卵白蛋白作为致敏原,腹腔注射BALB/c小鼠以建立Ⅰ型变态反应动物模型,并通过在卡介菌多糖核酸制剂效力实验中应用建立的小鼠模型来验证其可行性。结果  猪血清、牛血清白蛋白和卵白蛋白均可作为致敏原致敏BALB/c小鼠,但牛血清白蛋白和卵白蛋白的致敏效果优于猪血清。卡介菌多糖核酸制剂效力实验显示,牛血清白蛋白致敏小鼠建立的动物模型可更好地反映卡介菌多糖核酸制剂的效力。结论  牛血清白蛋白致敏小鼠建立的动物模型可用于卡介菌多糖核酸制剂效力实验。     

紫外光谱研究中药大黄有效成分与牛血清白蛋白的相互作用

目的　用紫外光谱研究大黄有效成分:大黄素、大黄酸和大黄酚与牛血清白蛋白的相互作用,了解大黄有效成分的生物结合活性。方法　采用紫外光谱法,通过测量加入牛血清白蛋白后药物吸光度值的变化,以Scatchard方程作图求解。结果　大黄三种主要有效成分:大黄素、大黄酸和大黄酚与牛血清白蛋白相互作用的结合常数分别为K=1.47×10⁵,K=5.00×10⁵,K=1.18×10⁴。其中大黄素与牛血清白蛋白的结合常数与文献值较一致,大黄酸、大黄酚与牛血清白蛋白的结合常数未见报道。结论　从结合机制上对测定结果进行了探讨,大黄类有效成分与牛血清白蛋白的结合能力随其所含极性基团的增多而增强。表明该方法研究药物蛋白相互作用简便、快速、可行。     

HPLC法测定胆酸类化合物与牛血清白蛋白的结合率

目的:研究胆酸类化合物与牛血清白蛋白的结合率,为进一步了解胆酸类化合物体内蛋白结合特性提供依据。方法:采用平衡透析法,以磷酸盐缓冲溶液为透析液,透析内液中加入1 mmol/L牛血清白蛋白1 ml,透析外液中加入质量浓度分别为2.02、1.01、0.51 mg/ml的胆酸和猪脱氧胆酸,振荡30 h后,以高效液相色谱法测定透析袋两侧磷酸盐缓冲溶液中胆酸类化合物的质量浓度,并计算胆酸类化合物与牛血清白蛋白的结合率。结果:3种质量浓度下,胆酸与牛血清白蛋白的结合率分别为78.7%、78.2%、59.4%,猪脱氧胆酸与牛血清白蛋白的结合率分别为68.9%、76.2%、81.8%。结论:胆酸类化合物与牛血清白蛋白具有中等强度的结合率,胆酸类化合物在体内不易因游离浓度过大而产生副作用。     

高效液相色谱法测定芹菜素在不同血浆中的蛋白结合率

目的:建立测定芹菜素在大鼠血浆、人血浆和牛血清白蛋白(BSA)中蛋白结合率的方法,并计算不同介质中的的相关参数。方法:采用高效液相色谱(HPLC)测定不同介质中芹菜素的浓度,并结合平衡透析法测定蛋白结合率。结果:高、中、低浓度下,芹菜素的血浆蛋白结合率分别为:大鼠血浆:(99.4±3.8)%、(99.6±5.6)%、(99.5±4.5)%;人血浆:(96.3±7.2)%、(97.9±10.5)%、(97.8±9.7)%;牛血清白蛋白:(98.7±8.6)%、(99.6±9.4)%、(99.1±8.0)%。结论:本方法快速、简便、可靠,芹菜素与大鼠血浆、人血浆和牛血清白蛋白结合率很高,且与血药浓度无显著相关性。     

用荧光参数表征牛血清白蛋白在脲中的变化过程

目的 研究牛血清白蛋白在脲中的构象变化过程,根据荧光参数的变化,建立蛋白构象与环境变化的关系,从分子水平探讨蛋白质构象变化而致不稳定的原因.方法 采用内源性荧光法、荧光淬灭法及荧光探针法研究牛血清白蛋白在脲中的构象变化过程.结果 随着脲浓度的增加,牛血清白蛋白中内源性色氨酸荧光峰位先蓝移后红移,荧光强度逐步衰减;而8 -苯胺基-1-萘磺酸与牛血清白蛋白结合物的荧光峰位则逐步红移,荧光强度逐渐衰减.结论 在变性过程中,牛血清白蛋白的色氨酸残基所处区域构象经历了一个先紧缩后舒展的三态过程;而8 -苯胺基-1-萘磺酸的探针结合位点与色氨酸残基可能位于牛血清白蛋白的不同区域,且构象变化更为灵敏.     

大黄素电化学性质的研究

目的:研究了大黄素的电化学性质以及大黄素与牛血清白蛋白(BSA)相互作用的电化学行为。方法:采用循环伏安法和差分脉冲伏安法对大黄素进行电化学行为的研究,并在不同pH值下进行线形扫描实验;同时应用紫外-可见分光光度法对大黄素与牛血清白蛋白的结合情况进行了研究。结果:大黄素在-0.522V处出现一个明显的还原峰;大黄素与牛血清白蛋白结合生成了一种非电活性的超分子化合物,并对结合反应的机理进行了探讨。结论:大黄素的还原峰电流与扫描速率呈简单线性关系。大黄素与牛血清白蛋白结合形成非电活性的超分子化合物导致大黄素氧化还原峰电流降低,峰电位基本不变,峰电流的下降值同所加入的BSA浓度在一定范围内呈线性关系。     

疫苗中残留卵清蛋白ELISA定量测定方法的建立

目的 建立ELISA法检测疫苗中残留卵清蛋白的含量.   方法 将进口纯化兔抗卵清蛋白抗体作为包被抗体,辣根过氧化物酶标记兔抗卵清蛋白抗体作为酶标抗体,以系列含量的卵清蛋白溶液为标准,采用ELISA双抗体夹心法,对供试品中残留卵清蛋白进行定量测定.   结果 最佳线性范围为1.25～20 ng/ml,相关系数r≥0.99;定量限度为1.25 ng/ml;准确性试验回收率为89.2%～103.6%;精密性试验内和试验间变异系数分别为5.6%～6.7%和4.2%～9.4%.与马血清、羊血清、人血清白蛋白、牛血清白蛋白、流行性感冒病毒裂解疫苗基质液及其他无残留卵清蛋白的疫苗均无交叉反应.   结论 本方法特异性强、灵敏度高,准确性、重复性和稳定性好,可用于疫苗中残留卵清蛋白的定量检测.     

Isolated rat kidney perfused with dextran and bovine serum albumin: a stable model for investigating renal drug handling

INTRODUCTION: The rat isolated perfused kidney (IPK) preparation is a very useful model for pharmacokinetic and pharmacologic studies. Bovine serum albumin (BSA) is the oncotic agent used most commonly in IPK models, but the protein is expensive and varies significantly in quality. The present study evaluated the use of dextran to replace a large proportion of BSA as the oncotic agent, to establish a more reliable and economic IPK model for pharmacokinetic studies. METHODS: The right kidneys of male Sprague-Dawley rats were isolated and perfused in recirculating mode with Krebs-Henseleit pH 7.4 buffer containing amino acids, glucose and 65 g/l BSA (BSA group, n=11) or 6.5 g/l BSA plus 36 g/l dextran (dextran/BSA group, n=6). (14)C-Inulin was added to the perfusate to permit estimation of glomerular filtration rate (GFR). An antiviral guanosine analogue, AM188, was administered to the IPK perfusate to investigate its renal disposition. During the 130-min experimental period, urine was collected in 10-min intervals and perfusate was collected at the midpoint of these intervals. RESULTS: The kidney functional parameters were generally better and more stable in the dextran/BSA IPKs when compared to the BSA group. At a similar perfusate flow rate, the IPKs in the dextran/BSA group exhibited lower renal artery perfusion pressure, a higher GFR, and more extensive tubular reabsorption of water, glucose, and sodium. These functional parameters were acceptable and stable throughout the whole experimental period in the dextran/BSA group. The renal clearance of AM188 was higher in the dextran/BSA group compared with that in the BSA group. DISCUSSION: Using a large proportion of dextran and a small proportion of BSA as oncotic agent in perfusate provides an improved IPK preparation. This offers a reliable and economic rat IPK model for pharmacokinetic studies.     

Influence of protein binding and metabolic interconversion on the disposition of sulfisoxazole and its N4-acetyl metabolite by the isolated perfused rat kidney

The renal clearances of sulfisoxazole (SX) and N4-acetylsulfisoxazole (NSX) were studied in the isolated perfused rat kidney (IPK). Studies were conducted with conventional bovine serum albumin perfusates as well as with dextran perfusates to assess the influence of perfusate protein binding on the disposition of these compounds by the IPK. The results presented herein indicate that the disposition of sulfisoxazole by the IPK involves both metabolism and excretion. The metabolism of SX to NSX is reversible and is influenced by protein binding since metabolism increased with increased free fraction (Ff). The excretion of SX and NSX reflects a complex interaction of filtration, secretion, and reabsorption. A comparison of clearance values between kidneys perfused with bovine serum albumin perfusate (Ff 0.1) and dextran perfusate (Ff 1.0) suggests that tubular secretion of SX is a function of total (unbound plus bound) rather than free (unbound) drug in the perfusate.     

Effect of plasma protein binding on the uptake of methadone and diazepam in the isolated perfused rat lung

The pulmonary uptake of the basic (pKa 9.1) lipophilic amine (methadone) and a nonbasic (pKa 3.4) lipophilic amine (diazepam) was compared in a single pass isolated perfused rat lung (IPL) preparation. The radiolabeled drugs were infused into the IPL for 10 min followed by a 30-min drug-free perfusion. If the perfusate (Krebs-Ringer bicarbonate buffer) contained 4.5% bovine serum albumin, the rapid and extensive uptake observed for methadone (32.4% of infused amount) was similar to that reported for other basic lipophilic amines. Uptake of the nonbasic diazepam was slight (3.4% of infused amount). These results are consistent with the idea that basic amines accumulate in lung tissues to a great extent. However, if the bovine serum albumin was omitted from the perfusate, diazepam uptake in the IPL increased about 10-fold while methadone uptake increased only slightly. This observation, together with the extensive binding of diazepam to plasma albumin, suggested that plasma protein binding was a major factor in limiting the pulmonary accumulation of this nonbasic lipophilic amine. Since many nonbasic drugs are known to have a high affinity for plasma albumin, the observed dependence of pulmonary drug accumulation on basicity of the amine may be related to the plasma protein binding as well as the characteristics of the interaction of amines with pulmonary tissue.     

适于研究药物相互作用的肝脏灌流模型

目的:改进和建立大鼠离体肝脏灌流模型.方法:采用Krebs-Henseleit 缓冲液(pH7.4)为港灌流液基液,含2%透析48h的牛血清白蛋白组分V、20%(V:V)洗过的人红细胞和0.3%葡萄糖.灌流速度1.5ml·min~(-1)·g~(-1),温度(37±0.5)℃,灌流压力1.7～1.33pKa.测定大鼠灌流过程中胆汁分泌量、耗氧量及灌流液pH、Na~ 、K~ 水平,并观察肝脏外观变化和组织切片的细胞形态学,评定肝脏功能.结果:本系统灌流中大鼠的各项考察指标正常,离体肝脏的存活力可达3h.结论:该模型适用于研究药物在肝脏代谢中的相互作用及其发生机制,也可用于研究某些药物特殊的代谢动力学特征.     

Placental Transfer of Diethylhexyl Phthalate in the Guinea-pig Placenta Perfused in Situ

Abstract: Placental transport of diethylhexyl phthalate (DEHP) in guinea-pigs has been studied with a placental perfusion technique. This transfer was shown to be independent of perfusion flow rate within 1.5–2.3 ml/min. when heparinized blood was used as perfusion media. When dextran solution (Macrodex^®) or dextran solution with the addition of Intralipid^® was used as perfusion medium there was no transport of DEHP from the maternal to the foetal compartment. The placental transport of DEHP was shown to be dependent on the albumin concentration in the perfusion media, up to an albumin concentration of 54 mg/ml. With γ-globulin solution as perfusion medium there was no placental transfer of DEHP.     

High-density lipoproteins decrease the biliary concentration of chlordecone in isolated perfused pig liver

Chlordecone (CD) is an organochlorine pesticide associated with albumin and high-density lipoproteins (HDL) in the plasma. It is found in higher concentrations in the liver than in other tissues and is excreted in the bile. The influence of plasma HDL on the biliary excretion of CD was studied using isolated pig liver perfused with a Krebs-Ringer bicarbonate solution containing albumin, dextrose, and pig red blood cells. Within 5 min after administration into the perfusion medium of [14C]CD bound to albumin or to HDL, only 13% of the [14C]CD dose remained in the perfusate, showing that CD is rapidly taken up by the liver. After 60 min the plasma concentration was constant at 0.008% dose/ml when [14C]CD was administered bound to albumin in the absence of HDL and at 0.004% dose/ml when administered bound to HDL. The mean concentration of CD in the bile was higher when CD was administered bound to albumin in the absence of HDL (0.039% dose/ml) than when it was administered bound to HDL (0.010% dose/ml). The elimination rate constant of CD from the liver into the bile was 0.007/min whether CD was administered bound to albumin or to HDL. The addition of HDL to the perfusion system after the administration of albumin-bound CD resulted in lower biliary CD concentrations. The results suggest that HDL affects the distribution of CD between the perfusate and liver and between liver and bile. In both cases, distribution toward the liver is favored.     

Influence of Albumin Concentration in the Foetal Circulation on the Placental Transfer of 2,2′,4,4′,5,5′-Hexachlorobiphenyl in the Guinea-pig

Abstract: Placental transport of 2,2′,4,4′,5,5′-hexachlorobiphenyl in the guinea-pig was studied by means of perfusion technique. This transfer was rapid when heparinized blood was used as perfusion medium. The same concentration of the chlorobiphenyl in both maternal and foetal compartments was achieved about 20 min. after start of infusion. It was also shown that when dextran solution or dextran solution with addition of Intralipid^® to the same concentration as the total lipid concentration in blood, were used as perfusion medium, there was no placental transport of the chlorobiphenyl. Furthermore, it was shown that the placental transport of the chlorobiphenyl was strongly dependent on the albumin concentration, but independent of the γ-globulin concentration in the perfusion medium.     

Role of unstirred water layer in the exsorption of quinidine

Intestinal ++exsorption of salicylic acid, thiopentone, theophylline, and quinidine was measured during perfusion of the intestinal lumen with Tyrode solution. The effect of pectin or bovine serum albumin added to the perfusate on intestinal clearance (CLi) was investigated. Increasing pectin concentration from 0.0 to 0.5, 1.0 and 1.5% gave CLi values for quinidine of 499 +/- 18, 363 +/- 35, 237 +/- 56, and 300 +/- 28 mL h-1 kg-1, respectively. One per cent of pectin in the perfusate also decreased the CLi of thiopentone, but had no effect on the CLi of salicylic acid or theophylline. Pectin may have increased the thickness of the unstirred water layer on the mucous membrane and the resistance of drug exsorption for some drugs. When bovine serum albumin was added, drug binding in the perfusate increased, and the CLi of salicylic acid, thiopentone, and theophylline increased; the CLi of quinidine was unaltered. Co-administration of theophylline with quinidine decreased the CLi of quinidine without affecting quinidine binding in serum or in the perfusate. The CLi theophylline was not affected by quinidine. These observations are consistent with the hypothesis that the exsorption of quinidine is rate-limited by diffusion through the unstirred water layer on the mucous membrane. The CLi of quinidine is affected by the microclimate-pH in the unstirred water layer. An alternative possibility is that quinidine exsorption is mediated by a carrier-transport pathway.     

Disposition Characteristics of Plasmid DNA in the Single-pass Rat Liver Perfusion System

Purpose. To define the hepatic uptake mechanism of a plasmid DNA, we quantitated the uptake of pCAT (plasmid DNA encoding chloramphenicol acetyltransferase reporter gene fused to simian virus 40 promoter), a model plasmid, after a single pass through the perfused rat liver using albumin- and erythrocyte-free Krebs-Ringer bicarbonate buffer (pH 7.4). Methods. [³²P]pCAT was introduced momentarily into this system from the portal vein as a bolus input or constant infusion mode, and the outflow patterns and hepatic uptake were evaluated using statistical moment analysis. Results. The venous outflow samples had electrophoretic bands similar to that of the standard pCAT, suggesting that the plasmid is fairly stable in the perfusate during liver perfusion. In bolus experiments, pCAT was largely taken up by the liver and the uptake was decreased with increase in injected dose. Statistical moment analysis against outflow patterns demonstrated that the apparent volume of distribution of pCAT was greater than that of human serum albumin, indicating a significant reversible interaction with the tissues. The results of collagenase perfusion experiments suggest that the hepatic accumulation of pCAT occurred preferentially in the nonparenchymal cells (NPC). The amount of total recovery in the liver decreased substantially by preceding administration of polyinosinic acid, dextran sulfate, succinylated bovine serum albumin, but not by polycytidylic acid. This suggests that pC AT is taken up by the liver via scavenger receptors for polyanions on the NPC. In constant infusion experiments, the presence of 2,4-dinitrophenol and NH₄C1 caused a significant increase in the outflow concentration of [³²P]pCAT and decrease by half in the total hepatic recovery than that of plasmid DNA administered alone, suggesting that plasmid DNA may undergo internalization by the NPC. Conclusions. The liver plays an important role in the elimination of plasmid DNA and a successful delivery system will be required to avoid its recognition by the scavenger receptors on the liver NPC.     

姜黄素白蛋白纳米粒的制备与评价

目的:优化姜黄素白蛋白纳米粒的处方工艺并对其特性进行表征。方法:以牛血清白蛋白为载体、通过反溶剂沉淀法制备载姜黄素纳米粒,以粒径为评价指标优选制剂处方及工艺,并考察所得纳米粒的放置稳定性、饱和溶解度及体外溶出度。结果:当有机相和水相体积之比为1∶20、药物与白蛋白用量之比为1∶1.5、保护剂为0.5%(V/V)甘露醇时,所得纳米粒冻干粉平均粒径约为320 nm。其在水、p H 6.8、p H 7.4 PBS中的溶解度显著高于原料药及物理共混物,体外溶出更快,且冻干粉的放置稳定性优于纳米混悬液。结论:白蛋白纳米粒处方能够改善姜黄素的水溶性及溶出度,放置稳定性较佳。