Computation of the effect of Donnan equilibrium on pH in equilibrium dialysis

In equilibrium dialysis, with a nondiffusible, charged protein on one side of the membrane, Donnan equilibrium leads to a pH difference across the membrane. Therefore, with an ionizable drug, the concentration dissolved in water will be different on the two sides of the membrane to an extent dependent on the pH difference and the pKa of the drug. This must be allowed for in calculating the concentration bound to protein. This paper develops, with certain restrictions, a method of calculating the pH difference when the solutions contain electrolytes, acids, including CO₂, and proteins. The method is applicable to pH differences across passive cell membranes.     

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.     

Binding of theophylline in human serum determined by ultrafiltration and equilibrium dialysis.

1 Binding of theophylline (80 mumol/l) was determined in serum from healthy subjects by equilibrium dialysis and by ultrafiltration, using [3H]-theophylline, at 22 degrees C and at different pH-values. pH was regulated by gassing with CO2 or by dialysing the serum against a phosphate buffer before use. 2 Binding of theophylline in serum was 34-38% determined by equilibrium dialysis and 41-45% determined by ultrafiltration at pH 7.4-7.5. The protein concentration in serum decreased by 12-16% during equilibrium dialysis and increased by 20% during ultrafiltration. The intersubject variation in binding was small. 3 Binding of theophylline in serum was pH-dependent with 25-30% bound at pH 7.0 and 58-60% bound at pH 8.1-8.3. Binding was significantly correlated to the fraction of ionized theophylline. 4 The binding of theophylline in normal human serum is about 35-40% at pH 7.4 and 22 degrees C. The difference in binding observed between equilibrium dialysis and ultrafiltration may be explained by the opposite changes in protein concentration during the experiment. 5 Control of pH is necessary to obtain physiologically relevant data on drug binding in serum.     

The role of buffer composition during equilibrium dialysis in determining concentrations of free diazepam in serum

Serum levels of free diazepam as determined with equilibrium dialysis depend on the composition of the dialyzing buffer with respect to Ca2+ and Cl-. Physiological concentrations of these ions double the obtained free serum levels, in line with data of diazepam containing albumin solutions. The diazepam binding capacity of serum was found to be higher than that of albumin alone, suggesting that albumin is probably not the only diazepam binding protein in serum. Evidence was also found that the diazepam binding to these other serum proteins is dependent on the presence of Ca2+ and Cl-.     

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.     

A pressure ultrafiltration method for serum free drug determination. Comparison with equilibrium dialysis using normal and uraemic sera

The clinical methods most frequently used for serum free drug determinations are equilibrium dialysis and ultrafiltration. Both methods have usually been considered equal when comparisons have been made on normal sera. The purpose of the study was to develop a reliable and rapid method for serum free drug determinations especially for disease states where there is an increased free fraction. This modification of the pressure ultrafiltration method proved to meet these demands. The method was tested in vitro by adding radiolabelled phenytoin in different concentrations to normal and uraemic sera. The within-run and between-run variation coefficients were both under 5%. In comparison with equilibrium dialysis the values obtained by the ultrafiltration method were 0.65% lower (p less than 0.001, Student's paired t-test) in normal and 0.37% lower (non-significant) in uraemic sera. There are some theoretical grounds to presume that the results obtained by means of the pressure ultrafiltration method would better reflect the real values than those obtained by means of equilibrium dialysis.     

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.     

Bronchial lability in allergic rhinitis.

The aim of this study was to assess the relationship between exercise bronchial lability and the changes in pulmonary function over a one year period in allergic rhinitis. Eighty four nonsmoking male medical students of whom twenty two were allergic rhinitics were studied. Pulmonary function status was significantly lower and exercise bronchial lability significantly higher in the allergics. Over a one year period the mean decline in the forced expiratory volume in one second (FEV1) was three times greater and in the forced expiratory flow rate in the middle half of the vital capacity (FEF 25-75%) 50% greater among the allergics. The change in FEV1 was positively related to the bronchial lability, indicating that greater bronchial lability was associated with less decline in pulmonary function in these subjects.     

Development of a high throughput equilibrium dialysis method

The identification of large numbers of biologically active chemical entities during high throughput screening (HTS) necessitates the incorporation of new strategies to identify compounds with drug-like properties early during the lead prioritization and development processes. One of the major steps in lead prioritization is an assessment of compound binding to plasma proteins, because it affects both the pharmacokinetics and pharmacodynamics of the compound in vivo. Equilibrium dialysis is the preferred method to determine the free drug fraction, because it is less susceptible to experimental artifacts. However, even low-volume standard equilibrium dialysis is currently not amenable to the HTS format. Those considerations dictate the development of a high throughput equilibrium dialysis device, without compromising the analytical quality of the data. The present paper demonstrates successful development of a 96-well format equilibrium dialysis plate. Plasma protein binding of three drugs, propranolol, paroxetine, and losartan, with low, intermediate, and high binding properties, respectively, were chosen for assay validation. The data indicate that the apparent free fraction obtained by this method correlates with the published values determined by the traditional equilibrium dialysis techniques.     

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).     

Impact of recovery on fraction unbound using equilibrium dialysis

Historically, recovery had been used to evaluate the data quality of plasma protein binding or tissue binding obtained from equilibrium dialysis assays. Low recovery was often indicative of high nonspecific binding, instability, or low solubility. This study showed that, when equilibrium was fully established in the dialysis assay, low recovery due to nonspecific binding had no impact on the determination of fraction unbound. The conclusion was supported by the principles of the equilibrium dialysis assay, experimental data, and mathematic simulations. The results suggested that the use of recovery as an acceptance criterion for the equilibrium dialysis assay in drug discovery was too restrictive, and introduced the additional burden of repeating studies unnecessarily.     

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).