马鞭草苷在不同血浆中蛋白结合率的HPLC测定

建立了HPLC法测定马鞭草苷在大鼠血浆、人血浆和牛血清白蛋白中的蛋白结合率,并计算不同种属血浆蛋白的相关参数.结果显示,体外马鞭草苷与大鼠血浆、人血浆和牛血清白蛋白结合率低,且与血药浓度无显著相关性.     

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。     

高效液相色谱法测定间尼索地平不同对映体在血浆中的蛋白结合率

目的:建立间尼索地平不同对映体在大鼠血浆、人血浆和牛血清白蛋白中蛋白结合率的测定方法,并计算不同种属血浆蛋白的相关参数。方法:采用平衡透析法测定蛋白结合率,用高效液相色谱法测定血浆中药物总浓度及游离药物浓度。结果:R-与S-间尼索地平的血浆蛋白结合率分别为:大鼠血浆为(97.4±8.2)%、(93.0±13.4)%;人血浆为(90.8±10.5)%、(89.2±9.9)%;牛血清白蛋白为(95.3±8.7)%、(91.0±5.7)%。最低定量下限为0.01ng。结论:在体外间尼索地平不同对映体与大鼠血浆、人血浆和牛血清白蛋白结合率很高,R-间尼索地平的结合率及蛋白结合药物的表观最大能力βp均较S-间尼索地平高。随着药物血浆浓度升高,R-间尼索地平结合率下降,S-间尼索地平结合率升高,均有一定的浓度依赖性。     

冬凌草甲素在不同种属血浆中蛋白结合率的HPLC法测定

目的:建立冬凌草甲素在大鼠血浆、人血浆和牛血清白蛋白中蛋白结合率的测定方法,并计算不同种属血浆蛋白的相关参数。方法:采用HPLC法测定血浆中药物总浓度及游离药物浓度,应用平衡透析法测定蛋白结合率。结果:大鼠血浆中冬凌草甲素高、中、低3个浓度的血浆蛋白结合率分别为(69.66±12.8)%,(59.62±12.6)%,(57.94±4.1)%;人血浆中冬凌草甲素高、中、低3个浓度的血浆蛋白结合率分别为(78.15±3.6)%,(77.92±8.8)%,(76.72±7.3)%;牛血清白蛋白中冬凌草甲素高、中、低3个浓度的血浆蛋白结合率分别为(35.58±7.2)%,(34.59±10.8)%,(32.03±6.0)%。结论:在体外冬凌草甲素与大鼠血浆、人血浆和牛血清白蛋白属中等结合型药物,且蛋白结合率随着药物血浆浓度的增加无明显的浓度依赖性。     

超滤法测定脱氢卡维丁的血浆蛋白结合率

目的:测定脱氢卡维丁的血浆蛋白结合率。方法:采用超滤法和高效液相色谱法对脱氢卡维丁的血浆蛋白结合率进行测定。结果:脱氢卡维丁与牛血清白蛋白、人血浆和大鼠血浆的蛋白结合率分别为(22.91±1.40)%、(54.35±2.95)%及(52.11±2.31)%。结论:脱氢卡维丁与血浆蛋白具有较低强度的结合。     

The Binding of Thiopental to Serum Proteins Determined by Ultrafiltration and Equilibrium Dialysis

Abstract: An ultra filtration method is described by which it is possible to estimate the protein bound fraction of a drug at its original concentration in a serum sample. The determination is carried out at 37° and pH 7.4. For thiopental no difference was found between the values of protein binding whether they were determined by the ultrafiltration method or by a less time consuming equilibrium dialysis against an equal volume of phosphate buffered isotonic sodium chloride solution. The equilibrium dialysis was used to measure the concentration of bound and unbound thiopental molecules; and the binding parameters in a two class binding model were determined. No evidence was found for binding to other proteins than albumin. About one binding site belonging to the first class was found per 1000 albumin molecules whereas an average of about 5 secondary sites were found for each albumin molecule. The association constants for the primary class of binding sites were 3.4×10⁶M^?1 for albumin and 13.3×10⁶M^?1 for serum while the values for the secondary class were 2.2 and 1.2×10³M^?1 for albumin and serum respectively. The estimates of association constants and number of binding sites were based on experiments with total thiopental concentrations ranging from 0.4 to 80 μg/ml. In a phosphate buffered albumin solution with 2 g albumin per 100 ml, a pH increase from 5 to 8 caused an increase in protein binding from 36 to 76%. A Scatchard plot using data from experiments with increasing albumin concentrations resulted in a “plot” with positive slope. The use of a curve like this is discussed and it is concluded that the binding parameters for thiopental are influenced by the albumin concentration.     

The binding of intravenous and oral biliary contrast agents to human and bovine serum albumin

Summary The binding of two homologous series of oral and intravenous biliary contrast agents to human and bovine serum albumin was investigated using the gel filtration technique. All intravenous compounds are bound to human serum albumin via one high affinity and several low affinity binding sites. Within the concentration range investigated, about 3–5 high affinity binding sites for the oral compounds were found on human serum albumin. In general, the intravenous compounds have a greater affinity for human serum albumin than the oral compounds. No significant differences were found for the binding of the oral compounds to human or bovine serum albumin, while the intravenous compounds have a higher affinity for bovine than for human serum albumin. The significance of the plasma protein binding of the biliary contrast agents for the hepatic uptake is discussed.     

Stereoselectivity and species difference in plasma protein binding of KE-298 and its metabolites.

In vitro protein binding of KE-298 and its plasma metabolites, deacetyl-KE-298 (M-1) and S-methyl-KE-298 (M-2), was high in rat (>97%), dog (>89%) and human plasma (>99%), respectively. Human serum albumin (>93%) was the main protein involved in the binding to plasma proteins, while the binding to human serum globulins was low (16-33%). The binding of KE-298 and its metabolites in all species of plasma was stereoselective. The (+)-(S)-enantiomers of these compounds bound rat, dog and human plasma proteins to a greater extent than did the (-)-(R)-enantiomers, except that the case of KE-298 was opposite in rat plasma. The stereoselective plasma levels of these compounds in rats, dogs, or humans would likely be due to stereoselective differences in binding to plasma albumin. The protein binding of M-1 in adjuvant-induced arthritis rat plasma was >97%, and the stereoselectivity was similar to the case of normal rat plasma. KE-298 and its metabolites remarkably displaced [14C]warfarin, which bound on albumin in a solution of diluted rat serum albumin. Similarly, there was a displacement of [14C]warfarin in solutions of dog and human serum albumin, and concomitantly the displacement of [14C]diazepam. [3H]Digitoxin was not displaced by any of the enantiomers in each albumin solution. No stereoselectivity was found in displacement by enantiomers of the three compounds. These results suggest that stereoselective protein binding can be attributed to quantitative differences in binding to albumin rather than to the different binding sites.     

Binding of [3H]mianserin to bovine serum albumin,human serum albumin and α1-acid glycoprotein

Scatchard plots which were curvilinear with negative slopes were obtained when the binding of [³H]mianserin to bovine serum albumin (BSA), human serum albumin (HSA), defatted human serum albumin (D-HSA) and α₁-acid glycoprotein (α₁-AGP) was studied with equilibrium dialysis with constant protein concentrations and various ligand concentrations. Binding parameters were estimated graphically and with a non-linear least-squares computer program, assuming two classes of independent binding sites. α₁-AGP had the highest binding affinity (K) and binding capacity (nK). The binding parameters, n and K were not independent of protein concentration when the BSA concentration was varied. Linear atypical Scatchard plots with positive slopes were obtained when the protein concentration was varied for BSA, HSA and D-HSA, at a fixed ligand concentration.     

Elucidating the relationship between acetazolamide plasma protein binding and renal clearance using an albumin infusion.

The effect of plasma protein binding changes on drug clearance is an important concept in clinical pharmacology. In a hypoalbuminemic patient receiving acetazolamide, albumin infusion (50 g) increased acetazolamide plasma protein binding towards normal as the serum albumin concentration rose (r = 0.91, P < .001). The ratio of acetazolamide renal plasma clearance to creatinine clearance decreased as serum albumin levels increased (r = 0.78, P < .05) and the unbound drug fraction fell (r = 0.88, P < .01), but clearance ratios based on unbound plasma acetazolamide levels did not change. Albumin infusion resulted in a nonparallel decline over time between plasma and unbound plasma acetazolamide concentrations. These data demonstrate that, over the range of observed serum albumin concentrations, acetazolamide renal plasma clearance is sensitive to changes in plasma protein binding. Furthermore, our findings emphasize the importance of measuring unbound drug levels when protein binding changes occur during the course of drug disposition studies. Finally, this methodology allows for the fascile assessment of the effects of plasma protein binding changes on renal drug clearance.     

Binding of perfluorooctanoic acid to rat and human plasma proteins

Perfluorooctanoic acid (PFOA) is a commercially important organic fluorochemical and is considered to have a long half-life in human blood. In this paper, PFOA binding to rat and human plasma proteins was investigated. On the basis of results from size-exclusion chromatography and ligand blotting, most PFOA was in protein-bound form in male and female rat plasma, and the primary PFOA binding protein in plasma was serum albumin. PFOA binding to rat serum albumin (RSA) in the gas phase was observed by electrospray ionization MS. (19)F NMR experiments revealed that binding to RSA caused peak broadening and chemical shift changes of PFOA resonances, and on the basis of this observation, the dissociation constant was determined to be approximately 0.3 mM. The dissociation constants for PFOA binding to RSA and human serum albumin (HSA) and the numbers of PFOA binding sites on RSA and HSA were also determined by a separation method using microdesalting columns. No significant difference was found between PFOA binding to RSA and PFOA binding to HSA. The dissociation constants for binding of PFOA to RSA or HSA and the numbers of PFOA binding sites were in the range of 0.3-0.4 mM and 6-9, respectively. On the basis of these binding parameters and the estimated plasma concentration of serum albumin, greater than 90% of PFOA would be bound to serum albumin in both rat and human blood.     

荧光猝灭法快速测定芦荟大黄素血浆蛋白结合率的研究

目的:建立荧光猝灭法快速测定芦荟大黄素血浆蛋白结合率的理论和实验方法学。方法:在模拟人体生理条件下,以牛血清白蛋白(BSA)为荧光剂,芦荟大黄素为荧光猝灭剂,研究激发波长280nm下的荧光光谱。结果:芦荟大黄素和BSA的荧光猝灭是静态猝灭过程,其结合常数K30℃=6.024×104 L.mol-1和K37℃=5.104×104 L.mol-1;结合分子数n30℃=0.9776和n37℃=1.0548;相互作用力主要是静电作用;同时建立了预测血浆蛋白结合率的理论模型,并根据实验数据分析了芦荟大黄素浓度与BSA浓度动态变化时药物血浆蛋白结合率的全程规律。结论:不同蛋白质浓度与药物浓度条件下血浆蛋白结合率处于动态变化中,血浆蛋白结合率随药物浓度的增大而减少。     

Peroxidase/hydrogen peroxide--or bone marrow homogenate/hydrogen peroxide--mediated activation of phenol and binding to protein

1. 14C-Phenol was metabolized by rat bone marrow homogenate and H2O2. The homogenate catalyst, however, was inactivated by preincubation with H2O2, presumably due to inactivation of the enzyme(s) involved in phenol metabolism. 2. The majority of the metabolized 14C-phenol was bound to bone marrow proteins. o,o'-Biphenol and p,p'-biphenol were the principal non-protein-bound products. Ascorbate was unable to remove phenol oxidation products bound to protein, although o,o'-biphenol recovery from the reaction mixture was markedly enhanced. Prior alkylation of protein thiols with N-ethylmaleimide decreased the binding of 14C-phenol oxidation products to bone marrow proteins by only 10-20%. 3. 14C-Phenol (200 microM) metabolism by horseradish peroxidase (10 micrograms) and H2O2 (200 microM) also resulted in extensive binding to externally added bovine serum albumin. The absorption spectrum of 14C-phenol oxidation products bound to bovine serum albumin was similar to that of bound oxidation products of o,o'-biphenol but not of p,p'-biphenol. 4. Protease digestion of bovine serum albumin bound 14C-phenol oxidation products, followed by ethyl acetate extraction, extracted 75% of the 14C, indicating that most of the binding is probably non-covalent. Up to 32% of the 14C-phenol oxidation products binding to bovine serum albumin may be covalent, since derivation with dinitrofluorobenzene and extraction under acid, but not alkaline, conditions extracted the 14C. The percentage of metabolites covalently bound to bovine serum albumin was increased to 59% when horseradish peroxidase concentration was decreased to 0.2 micrograms. 5. The thiol groups of bovine serum albumin were unaffected by o,o'-biphenol oxidation products, slightly decreased by phenol oxidation products, but were completely depleted by p,p'-biphenol oxidation products. 6. These results indicate that o,o'-biphenol oxidation products are responsible for much of the 14C-phenol binding to protein.     

HPLC法测定戟叶马鞭草苷在不同血浆中的蛋白结合率

目的:建立测定戟叶马鞭草苷在大鼠血浆、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相关参数。方法:采用高效液相色谱(HPLC)法测定不同介质中戟叶马鞭草苷的浓度,并结合平衡透析法测定其蛋白结合率。结果:低、中、高浓度下,戟叶马鞭草苷的蛋白结合率分别为大鼠血浆:(19.52±4.7)%、(25.60±5.4)%、(20.91±3.9)%;人血浆:(23.12±5.7)%、(21.76±6.5)%、(24.30±7.6)%;牛血清白蛋白:(25.83±7.0)%、(27.70±9.1)%、(26.19±5.6)%。结论:本方法快速、简便、可靠,戟叶马鞭草苷与大鼠血浆、人血浆和牛血清白蛋白的蛋白结合率低,且与血药浓度无显著相关性。     

Factors affecting in vitro protein binding of etoposide in humans.

Clinical studies have demonstrated that the plasma protein binding of etoposide, a widely used anticancer drug, is extensive (approximately 94%), highly variable among patients (10-fold range), and significantly related to serum albumin and total bilirubin concentration. The present study was designed to more thoroughly evaluate factors likely to affect etoposide protein binding under controlled in vitro conditions where single variables could be changed. Protein binding was determined using an equilibrium dialysis method with tritiated etoposide. The binding of etoposide was similar in serum or plasma, and heparin had no effect on binding. Etoposide binding decreased with increased pH, but no clinically significant difference was noted within the range of physiologic pH. Etoposide binding evaluated in single-source donor plasma was concentration-dependent over a concentration range of 1 to 250 micrograms/mL. Etoposide binding parameters determined in normal human plasma were characterized by a single class of binding sites of moderate affinity (K = 2.88 +/- 0.47 x 10(4)) and high capacity (nP = 5.07 +/- 0.5 x 10(-4); where n is the number of binding sites). The etoposide binding ratio was significantly correlated with albumin concentration (r2 = 99%, p less than 0.05). The characteristics of etoposide binding in a 4.0-g/dL solution of human serum albumin (K = 3.56 +/- 1.22 x 10(4) and nP = 5.58 +/- 0.16 x 10(-4)) suggest that the single class of binding sites is on albumin. Bilirubin caused a significant decrease in K, consistent with competitive binding, but only at higher bilirubin concentrations.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

齐墩果酸在人血浆蛋白和血清白蛋白中结合率的测定

齐墩果酸(oleanolic acid,OA)又名庆四素(图1),系五环三萜类化合物,以游离或结合成苷的形式广泛存在于白花蛇舌草、山楂、丁香、大枣、女贞子、枇杷叶、橡木和夏枯草等植物中。临床上主要用于急     

Study on glutathionesulfonic acid sodium salt as biodistribution promoter for thiopental sodium

The effects of glutathione (GSH) and glutathionesulfonic acid sodium salt [N-(N-gamma-L-glutamyl-L-beta-sulfoalanyl)glycine sodium salt, GSO3Na], which is a minor metabolite of GSH, on the pharmacokinetics of thiopental sodium were investigated in rats. The concomitant use of GSO3Na with thiopental sodium significantly increased the tissue-to-plasma concentration ratio (Kp) of thiopental sodium 60 min after its administration in the heart, lung, brain, liver, kidney, and spleen, while GSH did not affect them. On the other hand, the Kp value of thiopental sodium 5 min after its administration with concomitant GSO3Na decreased significantly only in the spleen. Neither GSO3Na nor GSH changes the pharmacokinetic parameters of thiopental sodium. Significant change of the binding ratio of thiopental sodium to bovine serum albumin (BSA) was not observed by the addition of less than 5-fold GSO3Na. About 50% of thiopental sodium was bound to the brain, lung or liver, however, no significant change of this binding ratio was observed by the concomitant use of GSO3Na. The partition coefficient of thiopental sodium apparently increased by the concomitant use of GSO3Na but not by GSH. This phenomenon seemed to be concerned with a mechanism to increase the Kp values of thiopental sodium in the tissues. The increment in the drug distribution to tissues with concomitant GSO3Na observed in this study is useful information for the application of drug combinations as a biodistribution promoter.     

Impact of protein binding on the availability and cytotoxic potency of organochlorine pesticides and chlorophenols in vitro

In vitro toxicity data are generally based on nominal concentrations and thus depend on both activity and availability of a compound. The aims of the present study were to examine the influence of protein binding on the cytotoxicity of selected organochlorine pesticides and chlorophenols in Balb/c 3T3 cell cultures and to determine parameters of protein binding which can be used to estimate protein bound fractions and to model distribution in vitro. EC(50)-values derived from concentration-effect relationships determined in the presence of various concentrations of bovine serum albumin (BSA) were linearly correlated to BSA concentration. Increasing the BSA concentration from about 1.2 to 40 mg/ml increased the EC(50)-values by factors between 3.4 and 34.4. Molar ratios of substance bound to albumin ranged from 0.11 to 2.42. Calculated fractions bound to albumin in the normal growth medium were 0.075-0.17 (p,p'-DDT, p,p'-DDE, dieldrin, lindane), 0.09-0.1 (4-mono- and 2,4-dichlorophenol), 0.68 (2,4,5-trichlorphenol) and almost 1.0 (pentachlorophenol). At 40 mg/ml BSA any compound was largely bound to albumin (fractions bound > or = 0.74). Distribution modelling revealed that the availability of the highly hydrophobic organochlorines additionally was significantly reduced by partitioning into lipids. The results clearly demonstrate that nominal and relative toxic potencies of organochlorine pesticides and chlorophenols determined in vitro are substantially influenced by effects of protein binding on availability.     

Effect of protein concentration on the binding of drugs to human serum albumin--I. Sulfadiazine, salicylate and phenylbutazone.

The binding of sulfadiazine, salicylate or phenylbutazone to human plasma and human serum albumin has been studied as a function of protein concentration. Results show that, as the concentration of albumin increases, the binding of all three drugs decreases. This phenomenon does not appear to be due to the presence of endogenous-competing ligands in serum or crystalline albumin preparations. Fluorescence depolarization measurements of dansylglycine complexes with human serum albumin show no evidence for the “molecular aggregation” of human serum albumin at high concentrations. Binding parameters obtained with dilute albumin solutions when extrapolated to physiological albumin concentrations may predict a higher degree of binding than that which is observed by direct measurement. The phannacokinetic implications of these findings are discussed.