Fluid shift and protein leakage corrections in protein binding equilibrium dialysis experiments

Conventional equilibrium dialysis methods used in the estimation of ligand-macromolecule binding parameters (affinities, capacities. unbound ligand fractions, etc.) tacitly assume and require that no ligand-macromolecule binding occurs on the buffer side of the dialysis chamber. Unfortunately, this is almost never a valid assumption, as small amounts of plasma proteins are invariably detected in the buffer chamber. Provided the extent of protein leakage is in the usual region (about 0.1%) and the extent of ligand binding does not exceed approximately 99%. errors associated with conventional free fraction estimations obtained from calculations ignoring the effect of protein leakage are usually small (about 1–10% error). As ligand binding exceeds 99%, significant errors may ensue. A generalized theoretical equilibrium dialysis method is developed which permits the estimation of association constants, the number of binding sites and free fraction determinations for a model employing any number of classes of binding sites. Application of the method requires a minimum of two experimental runs for each class of binding sites; volume shifts are automatically adjusted for by the method.     

Protein binding of glufosinate and factors affecting it revealed by an equilibrium dialysis technique

We investigated the protein binding of glufosinate ammonium (GLF) and several factors affecting this binding using human serum albumin (HSA) and human volunteer serum under various conditions. The mean ratios of the free GLF (RFr-GLF) to 4% HSA were examined in the sera of patients described elsewhere at GLF levels from 1 microg/mL to 500 microg/mL; the range was found to be only from 0.80 to 0.88. Neither the incubation temperature nor buffers containing different chloride ion concentrations had any effect on the RFr-GLF to HSA. Moreover, the addition of heparin, glycoprotein-alpha1-acid (AAG), and sodium azide had no effect on the RFr-GLF. However, pH of the isotonic phosphate buffer and the addition of palmitic or oleic acid were seen to have an effect. In this study, the mean RFr-GLF to healthy human serum was 0.99. This high value was evidenced that GLF was rapidly excreted through the renal route.     

Impact of pH on plasma protein binding in equilibrium dialysis

Many pharmacokinetic analyses require unbound plasma concentrations, including prediction of clearance, volume of distribution, drug-drug interactions, brain uptake analysis, etc. It is most often more convenient to measure the total drug concentration in plasma rather than the unbound drug concentration. To arrive at unbound plasma concentrations, separate in vitro determinations of the plasma protein binding of a drug are usually carried out in serum or in plasma, and the plasma pharmacokinetic results are then mathematically adjusted by this fraction unbound ( f u,p). Plasma protein binding or the drug fraction unbound in plasma ( f u,p) is known to be affected by protein, drug, free fatty acid concentrations, lipoprotein partitioning, temperature, pH, and the presence or absence of other drugs/displacing agents within plasma samples. Errors in f u,p determination caused by lack of adequate pH control in newer assay formats for plasma protein binding (e.g., 96-well equilibrium thin walled polypropylene dialysis plates) will have significant drug-specific impact on these pharmacokinetic calculations. Using a diverse set of 55 drugs and a 96-well equilibrium dialysis plate format, the effect of variable pH during equilibrium dialysis experiments on measured values of f u,p was examined. Equilibrium dialysis of human plasma against Dulbecco's phosphate buffered saline at 37 degrees C under an air or 10% CO 2 atmosphere for 22 h resulted in a final pH of approximately 8.7 and 7.4, respectively. The ratio of f u,p at pH 7.4 (10% CO 2) vs pH 8.7 (air) was >or=2.0 for 40% of the 55 compounds tested. Only one of the 55 compounds tested had a ratio <0.9. Select compounds were further examined in rat and dog plasma. In addition, physicochemical properties were calculated for all compounds using ACD/Labs software or Merck in-house software and compared to plasma protein binding results. Changes in plasma protein binding due to pH increases which occurred during the equilibrium dialysis experiment were not species specific but were drug-specific, though nonpolar, cationic compounds had a higher likely hood of displaying pH-dependent binding. These studies underscore the importance of effectively controlling pH in plasma protein binding studies.     

Factors affecting the measurement of lidocaine protein binding by equilibrium dialysis in human serum

A systematic study was undertaken to assess in vitro factors that influence the value of the lidocaine free fraction obtained by equilibrium dialysis in human serum. These factors include pH readjustment to 7.40 after serum storage; choice of buffers for dialysis; the effect of phosphate buffer ionic strength; temperature of storage for serum samples; the use of untreated versus silanized glassware for storage; and age of serum. It was concluded that the pH of serum that contains lidocaine must be brought back to the original whole blood pH found in the patient before equilibrium dialysis because the protein binding of lidocaine is critically dependent on pH. It was also found that Krebs-Ringer bicarbonate buffer, when used with room air atmosphere in the dialysis cell, is not adequate to control pH even when serum pH is readjusted to the physiological pH of the patient. Isotonic phosphate buffer and 0.10 M phosphate buffer are effective for pH control and give identical values of lidocaine free fraction when the original serum sample is first pH-adjusted. If the pH of the serum is correct and the pH of the buffer remains constant, then freezing, the choice of container, or the age of serum are not important variables affecting the measurement of the lidocaine free fraction.     

The protein binding of timegadine determined by equilibrium dialysis.

The protein binding of timegadine to albumin, serum, plasma and plasma enriched with the acute phase reactants alpha 1-acid glycoprotein, alpha 1-anti-trypsin and C-reactive protein was determined by equilibrium dialysis. The effects of other analgesic and anti-inflammatories (indomethacin, ketoprofen, paracetamol and sodium salicylate) and other basic drugs (disopyramide, lignocaine, propranolol and quinidine) on the binding of timegadine were also determined. Timegadine binding was concentration-dependent up to 0.5 micrograms/ml, but independent above this level up to 10.0 micrograms/ml, the mean and standard error being 93.8 +/- 0.5%. Albumin accounted for only 32.4% of timegadine bound to plasma. Plasma enrichment with the acute phase reactants led to significant increases in timegadine binding. Simultaneous dialysis with other drugs caused significant decreases in timegadine binding.     

Validation of a rapid equilibrium dialysis approach for the measurement of plasma protein binding

Equilibrium dialysis (ED) is one of the most frequently used approaches to investigate drug binding, where the major drawbacks are the time to reach equilibrium (varying between 6 and 24 h), a long assay preparation time and complexity of automation. A rapid equilibrium dialysis (RED) device has recently become commercially available (Pierce Biotechnology, ThermoFisher Scientific, Waltham, MA) offering the potential for reduced preparation and equilibration times. The RED device comprises a Teflon base plate which holds up to 48 disposable dialysis cells. Each dialysis insert is made up of two side-by-side chambers separated by a vertical cylinder of dialysis membrane with a high membrane surface area-to-volume ratio. An independent validation of the RED approach for the measurement of human plasma protein binding (PPB) was carried out as a comparative analysis with standard ED evaluating equilibration time, assay reproducibility and accuracy and ease of use. Using a diverse set of 18 commercially available drugs spanning a range of physicochemical properties we have shown this to be a robust and accurate methodology, with a shorter preparation and dialysis time, capable of being automated as a high-throughput assay for the determination of PPB.     

Nonlinear plasma protein binding and haemodialysis clearance of prednisolone

Summary The impact of nonlinear plasma protein binding of a drug on its removal by haemodialysis has been quantified. Prednisolone 10–100 mg was given i.v. to 10 renal transplant patients on haemodialysis for acute tubular necrosis. Dialysate and afferent and efferent blood samples were collected simultaneously in 67 instances. Total and unbound prednisolone in plasma and its total concentration in blood and dialysate were assessed by high performance liquid chromatography and equilibrium dialysis. The amount of prednisolone lost, as measured directly in the dialysate (21.8±4.4 µg/min, ± SE), was predictable from the afferent-efferent blood concentration differences (20.1±4.8 µg/min), but not from measurements of total afferent-efferent prednisolone concentrations in plasma (13.1±3.0 µg/min). The amount of prednisolone lost in the dialysate increased linearly with unbound (r ²=0.96) and hyperbolically with the total prednisolone concentration in plasma. The latter hyperbolic relationship is adequately described by the equation for nonlinear plasma protein binding, using the affinity and capacity constants of albumin and transcortin for prednisolone (r ²=0.98). Thus, the haemodialysis clearance of total prednisolone is concentration-dependent, while the clearance of unbound prednisolone is constant (76 ml/min). Free clearance values or measurements of afferent-efferent blood concentrations are mandatory for a drug showing nonlinear plasma protein binding in order to predict the amount lost in the dialysate.Abbreviations Used C_Ba Afferent blood concentration [ng/ml] - C_Be Efferent blood concentration [ng/ml] - C_D Dialysate concentration [ng/ml] - C_Pa Afferent plasma concentration [ng/ml] - C_PaFree Free concentration of prednisolone in afferent plasma [ng/ml] - C_Pe Efferent plasma concentration [ng/ml] - CAP_A Binding capacity of albumin [µg%] - CAP_T Binding capacity of transcortin [µg%] - Cl_B Blood clearance [ml/min] - Cl_Free Free plasma clearance [ml/min] - Cl_P Plasma clearance [ml/min] - H_a Haematocrit in afferent blood - H_e Haematocrit in efferent blood - K Partition coefficient between red blood cells and plasma - KD_A Dissociation constant of albumin (M/L) - KD_T Dissociation constant of transcortin (M/L) - Q_B Blood flow [ml/min] - Q_D Dialysate flow [ml/min] - Q_F Flow of ultrafiltrate [ml/min] - t₁ First sampling time point after haemodialysis started - t₂ Last sampling time point before haemodialysis was discontinued - Z_Ddi Loss of prednisolone in the dialysate by diffusion [µg/min] (Z indicates calculated for each sampling time point) - _Ddi Mean loss of prednisolone in the dialysate by diffusion [µg/min] ( indicates calculated for the entire haemodialysis period) - Z_Dtot Total amount of prednisolone recovered in the dialysate [µg/min] - _Dtot Mean total amount of prednisolone recovered in the dialysate [µg/min] - Z_Du Loss of prednisolone in the dialysate by ultrafiltration [µg/min] - _Du Mean loss of prednisolone in the dialysate by ultrafiltration [µg/min] - Z_cB Amount of prednisolone lost in the dialysate calculated from afferent-efferent blood concentrations [µg/min] - _cB Mean amount of prednisolone lost in the dialysate calculated from afferent-efferent blood concentrations [µg/min] - Z_cP Amount of prednisolone lost in the dialysate calculated from afferent-efferent plasma concentrations [µg/min] - _cP Mean amount of prednisolone lost in the dialysate calculated from afferent-efferent plasma concentrations [µg/min] - Z_cPK Amount of prednisolone lost in the dialysate calculated from afferent-efferent plasma concentrations corrected by the partition coefficient [µg/min] - _cPK Mean amount of prednisolone lost in the dialysate calculated from afferent-efferent plasma concentrations corrected for the partition coefficient [µg/min] Abstract presented to the American Society of Nephrology, Washington, 1981     

Development and validation of a 96-well equilibrium dialysis apparatus for measuring plasma protein binding

A 96-well equilibrium dialysis block was designed and constructed that is compatible with most standard 96-well format laboratory supplies and instruments. The unique design of the dialysis apparatus allows one to dispense and aspirate from either or both the sample and dialysate sides from the top of the apparatus, which is not possible with systems currently on the market. This feature permits the investigator to analyze a large number of samples, time points, or replicates in the same experiment. The novel alignment of the dialysis membrane vertically in the well maximizes the surface-to-volume ratio, eliminates problems associated with trapped air pockets, and allows one to add or remove samples independently or all at once. Furthermore, the design of the apparatus allows both the sample and dialysate sides of the dialysis well to be accessible by robotic systems, so assays can be readily automated. Teflon construction is used to minimize nonspecific binding of test samples to the apparatus. The device is reusable, easily assembled, and can be shaken in controlled temperature environments to decrease the time required to reach equilibrium as well as facilitate dissolution of test compounds. Plasma protein binding values obtained for 10 diverse compounds using standard dialysis equipment and the 96-well dialysis block validates this method.     

平衡透析法测定人体外血浆中原花青素B2、表儿茶素蛋白结合率

目的 建立检测原花青素B2、表儿茶素在血浆中药物浓度的方法,并测定其体外血浆蛋白结合率.方法 采用平衡透析法测定血浆蛋白结合率,建立HPLC法测定各成分的血药浓度及游离药物浓度,生物样本用乙酸乙酯溶液提取的方法.结果 在低、中、高3种浓度下,原花青素B2和表儿茶素在人体外血浆中的蛋白结合率分别为(44.21±2.80)%,(52.60±1.92)%,(48.11±3.09)%和(47.12±2.85)%,(55.03±2.47)%,(43.69±1.53)%.结论 采用HPLC法对原花青素B2、表儿茶素进行分离,方法简便、可靠、稳定.体外实验中原花青素B2和表儿茶素与人血浆属中等结合型药物,且蛋白结合率随着药物浓度的增加无明显的浓度依赖性.     

In vitro plasma protein binding determination of flunarizine using equilibrium dialysis and liquid chromatography-tandem mass spectrometry

A highly sensitive method was developed and validated for determining the free fraction of flunarizine in human plasma. Equilibrium dialysis was used for the separation of free (unbound) drug and liquid chromatography/tandem mass spectrometry (LC-MS/MS) was used for quantitation. Post-dialysis plasma or buffer samples of 0.2 mL were extracted using a liquid-liquid extraction procedure and analyzed using a high performance liquid chromatography electrospray tandem mass spectrometer system. The compounds were eluted isocratically on a Supelco Supelcosil ABZ+Plus column, ionized using a positive ion atmospheric pressure electrospray ionization source, and analyzed using multiple reaction monitoring. The ion transitions monitored were m/z 405-->203 for flunarizine and m/z 409-->207 for flunarizine-d4 (internal standard, IS). The chromatographic run time was 3.5 min per injection, with retention times of 2.1 min for both flunarizine and IS. The calibration curve for flunarizine was linear over the concentration range of 0.25-2000 ng/mL (r(2)>0.9989) in the combined matrix of human plasma and isotonic sodium phosphate buffer (1:1, v/v) with the lower limit of quantitation of 0.25 ng/mL. The inter-assay coefficient of variability (CV) for the quality control samples was less than 13.5%, and the inter-assay percent nominal was greater than 98.2%. In vitro protein binding of flunarizine was determined at concentrations of 5, 10 and 100 microg/mL using the validated method. Flunarizine was extensively bound to plasma protein with a 0.083+/-0.005% overall percent free drug in plasma and a CV value less than 7.8%. This validated method will be used for the ex vivo assessment of flunarizine protein binding in human plasma from a drug-drug interaction clinical study.     

Improved procedure for the determination of protein binding by conventional equilibrium dialysis

The binding of drugs to plasma proteins has been studied extensively using a variety of methods, including equilibrium dialysis. Published information on controls used in these studies is frequently inadequate; in other cases, there are deficiencies in the experimental design for the controls. A method is described that eliminates many of the problems associated with artifactual errors in dialysis studies. Multiple replicated controls are performed at the same time as the test, under identical conditions. The controls are used to correct for concentration-dependent binding of drug to the membrane or other equipment. The method was used to determine the binding of sulfadimethoxine to CF-IV-1 alpha-globulin at therapeutic concentrations. The level of binding was low (9-13%), but the stringent control technique permitted statistical analysis which showed each mean test value to be significantly different from its corresponding control. Furthermore, there was a linear relationship between the control-corrected percentage binding values and total drug concentration, whereas there was no correlation between total drug concentration and the uncorrected percentage binding values.     

Influence of volume shifts on drug binding during equilibrium dialysis: Correction and attenuation

A time-dependent volume shift from buffer to plasma, which occurs during equilibrium dialysis, decreased the protein binding of disopyramide and its capacity constant, and had no effect on the binding association constant. The volume-dependent decrease in disopyramidine binding may be corrected for by use of a derived equation. Inclusion of dextran, 2.5% (w/v), and use of a thick, low molecular weight cutoff membrane was the most effective technique in attenuating the volume shift. The plasma (serum) protein binding of the basic drugs lidocaine, disopyramide,propranolol, and diazepam was decreased when protein was diluted to 88 % or less of its undiluted concentration as a consequence of the volume shift. The protein binding of clofibrate, a highly bound acid drug, was more sensitive to volume shifts than the four basic drugs. Correction of drug binding for volume shifts was reasonably successful for most drugs. The highest binding measured for all drugs was associated with the lowest volume shift.     

平衡透析并液质联用法同时测定度洛西汀、氟西汀和阿戈美拉汀的人血浆蛋白结合率

目的 建立平衡透析并液质联用法(HPLC-MS/MS),同时测定度洛西汀、氟西汀和阿戈美拉汀在人血浆中的蛋白结合率.方法 用平衡透析法处理血浆得透析内液和透析外液.以地西泮作为内标,色谱柱为WATERS Xterra(R) RP18(4.6 mm×100 mm,3.5 μm),以甲醇-水(5 mmol·L-1醋酸铵-0.3%甲酸)为流动相,梯度洗脱,用电喷雾离子源,正离子多反应监测.考察该方法的特异性、标准曲线与最低定量限、精密度与回收率、基质效应以及稳定性. 结果 在血浆和磷酸盐缓冲液(PBS)中,度洛西汀、氟西汀和阿戈美拉汀的标准曲线线性良好,批内、批间精密度均<15%,提取回收率85.89%~106.86%,内标归一化基质效应在86.55%~104.94%,且稳定性均较好. 结论 该方法准确、灵敏、简便,可用于度洛西汀、氟西汀和阿戈美拉汀的人血浆蛋白结合率的研究.     

Plasma protein binding of phenol in dogs and rats as determined by equilibrium dialysis and ultrafiltration

Plasma-protein binding of phenol was examined in rats with and without ether anesthesia. Ether anesthesia decreased the percentage of phenol bound by rat plasma. The phenol binding capacities of dog and rat plasma were compared using equilibrium dialysis and ultrafiltration. For the wide range of phenol concentrations studied, dog plasma had a consistently higher binding capacity than rat plasma as demonstrated by both techniques. Higher binding was obtained by ultrafiltration than by equilibrium dialysis in both dog and rat plasma. Dog and rat serum albumins were responsible for 72–73% of the phenol binding in plasma, while bovine globulins either did not bind phenol or had only minimal binding capacities.     

Evaluation of equilibrium dialysis volume shifts: A comment

Previous authors have presented in this journal an equation defining the fractional shift in volume (f_s)as a general equation implying applicability to a wide variety of circumstances. The equation concerned actually applies only when the starting volumes before dialysis are equal. A better general definition is given by f_s=(volume shift)/(starting volume of protein solution).     

Evaluation of equilibrium dialysis volume shifts: a comment

Previous authors have presented in this journal an equation defining the fractional shift in volume (fs) as a general equation implying applicability to a wide variety of circumstances. The equation concerned actually applies only when the starting volumes before dialysis are equal. A better general definition is given by fs = (volume shift)/(starting volume of protein solution).     

Studies of drug binding to plasma proteins using a variant of equilibrium dialysis

The plasma protein binding of three model compounds was investigated using a variant of equilibrium dialysis, denoted comparative equilibrium dialysis (CED), and the results were compared with those obtained with ultrafiltration (UF). In CED, the buffer that the plasma is dialysed against in traditional equilibrium dialysis is replaced by, for example, plasma from other species. The CED method has the advantage that the unbound concentration (C(u)) does not need to be measured, which can be difficult for drugs with extremely small unbound fractions. Instead, the ratio of the total drug concentration (C(tot)) on either side of the dialysis membrane at equilibrium is a direct measure of the relative binding properties of the two plasma types. For the first model compound, having an unbound fraction (f(u)) of about 0.05% in human plasma, the time to reach equilibrium was too long (> or =40 h) to make the CED technique feasible in practice. For the second model compound, the more weakly bound drug NAD-299 (with an unbound fraction of about 2% in human plasma), the CED equilibration times were considerably shortened (< or =16 h), and the technique was applied to plasma from three different species. Large discrepancies between the CED and UF results were seen, CED always giving rise to much lower C(tot) differences than expected from the UF results. It is suspected that this discrepancy was due to equilibration between the dialysis chambers of all plasma components with a molecular weight less than the cut-off of the membrane. This equilibration causes altered binding properties compared to the initial plasma. When performing ultrafiltration on plasma where drug was added to untreated plasma or added to blank plasma that was equilibrated against plasma from the same or from another species, the change of binding properties was confirmed. To ensure that the results were not specific for NAD-299, a third model compound, tolterodine, was also included. The same trends as for NAD-299 were seen. Because of the long equilibration times for compounds with high protein binding and, in particular, the suspected partial mixture of low molecular weight compounds from the two plasma types and the subsequent change of binding properties, we cannot recommend the CED method as a tool for studying relative protein binding.     

Factors influencing the protein binding of azosemide using an equilibrium dialysis technique

Various factors most likely to influence the plasma protein binding of azosemide to 4% human serum albumin (HSA) were evaluated using equilibrium dialysis at the initial azosemide concentration of 10 μg mL^?1. It took approximately 8h of incubation to reach an equilibrium between 4% HSA and isotonic phosphate buffer of pH 7.4 containing 3% dextran (the ‘buffer’) using a Spectra/Por 2 membrane (molecular weight cut-off 12000–14000) in a water bath shaker kept at 37°C and a rate of 50 oscillations min^?1. Azosemide was fairly stable both in 4% HSA and in the ‘buffer’ for up to 24h. The binding of azosemide to 4% HSA was constant (95.5 ± 0.142%) at azosemide concentrations ranging from 5 to 100 μg mL^?1. However, the extent of binding was dependent on HSA concentration: the values were 88.4, 91.0, 92.2, 94.2, 94.9, 94.9, and 94.9% at albumin concentrations of 0.5, 1, 2, 3, 4, 5, and 6% respectively. The binding was also dependent on incubation temperature; the binding values were 97.0, 94.9, and 94.9% when incubated at 6, 28, and 37°C, respectively. The binding of azosemide was also influenced by buffers containing various chloride ion concentrations and buffer pHs. The binding values were 95.3, 94.9, and 93.6% for the chloride ion concentrations of 0, 0.249, and 0.546%, respectively, and the unbound values were 6.8, 5.1, 3.8, 3.4, and 3.3% for buffer pHs of 5.8, 6.4, 7.0, 7.4, and 8.0, respectively. The binding of azosemide was independent of the quantity of heparin (up to 40 UmL^?1), AAG (up to 0.16%), sodium azide (NaN₃, up to 5%), its metabolite, Ml (up to 10 μg mL^?1), and anticoagulants (EDTA and citrate).     

平衡透析法研究荭草及其制剂的人血浆蛋白结合率

目的:比较荭草提取物及其复方制剂中两类成分的人血浆蛋白结合率。方法:以超高效液相色谱—质谱联用为检测手段,结合平衡透析法考察单味荭草提取物及注射用复方荭草在人血浆中的蛋白结合率并计算相关参数。结果:在所研究浓度范围内,原儿茶酸与血浆蛋白具有中等强度结合,异荭草素与血浆蛋白结合力较强。经统计分析,两者与血浆蛋白的结合能力在考察浓度范围无显著差异;其中异荭草素指标在复方中的血浆蛋白结合率有所升高,复方中其他的血浆蛋白结合动力学参数相对单味荭草也有一定变化。结论:荭草中化学成分的血浆蛋白结合无浓度依赖性,而在组成复方后某些成分的结合率可能发生改变。     

Factors affecting ceftriaxone plasma protein binding during open heart surgery

Factors most likely contributing to reduced ceftriaxone plasma protein binding in patients undergoing open heart surgery (OHS) were examined. Binding was determined by equilibrium dialysis. It was found that ceftriaxone does not bind significantly to red blood cells, alpha 1-acid glycoprotein, or to protamine, and that the pH of serum did not significantly affect binding. Albumin is the major protein to bind ceftriaxone, and binding decreases with lower albumin concentrations due to fewer binding sites. The binding of ceftriaxone was not affected by the in vitro addition of heparin or methylprednisolone, but high concentrations of methylprednisolone hemisuccinate increased the free fraction of ceftriaxone. Increased concentrations of free fatty acids (FFA) were demonstrated in several patients undergoing OHS. The in vitro addition of palmitic, stearic, linoleic, and oleic acids in high concentrations decreased the binding of ceftriaxone. Ceftriaxone binding in patient samples correlated with the molar ratio of FFA to albumin, but not to either individually. The dual effect of increased FFA and decreased albumin concentrations in OHS patients appears responsible for most of the observed binding alterations.