Equilibrium dialysis-ultrafiltration method

In the equilibrium dialysis/ultrafiltration method for determining free testosterone in blood, a sample is first equilibrated with radioactive testosterone. Free steroid is then separated from bound steroid by filtration through an anisotropic, hydrophilic ultrafiitration membrane. The driving force for ultrafiltration is provided by centrifugation at 1000 to 2000 xg. Filtrate containing free steroid collects in the filtrate cup, whereas protein-bound steroid remains above the filter. Radioactivity in the filtrate is a measure of... 

Norris RLG, Ahokas JT, Ravenscroft PJ (1982) Determination of unbound fraction of disopyramide in plasma a comparison of equilibrium dialysis, ultrafiltration through dialysis membranes and ultrafree anticonvulsant drug filters. J Pharmacol Methods 7(1) 7—14... 

The most commonly used methods to determine drug binding to plasma proteins are equilibrium dialysis, ultrafiltration, and microdialysis. All have advantages and disadvantages, and results are method- and condition-specific. 

A variety of techniques are available for protein binding measurement including equilibrium dialysis, ultrafiltration, spectrofluorometric, and microdialysis methods. 

Three methods were used in this research to measure the extent of binding of organic pollutants to dissolved humic materials. They were equilibrium dialysis, solubility measurements and changes in sorption behavior in the presence of humic materials. Other authors have used solubility measurements, ultrafiltration and volatilization measurements. The methods will be described in the following paragraphs. 

Several variants of separation methods based on dialysis, ultrafiltration, and size exclusion chromatography have been developed that work under equilibrium conditions. Size exclusion chromatography especially has become the method of choice for binding measurements. The Hummel-Dreyer method, the vacancy peak method, and frontal analysis are variants that also apply to capillary electrophoresis. In comparison to chromatographic methods, capillary electrophoresis is faster, needs only minimal amounts of substances, and contains no stationary phase that may absorb parts of the equilibrium mixture or must be pre-equilibrated. 

A number of experimental methods are used to determine the amount of bound drug and/or free drug in a mixture, including equilibrium dialysis, steady-state dialysis, ultrafiltration, and size exclusion chromatography. Each of the methods is described briefly here. 

From the great variety of methods for the determination of protein binding three separation methods, equilibrium dialysis (ED), ultrafiltration (UF), and ultracentrifugation (UC) and a non-conventional method with the binding to immobilized proteins has been chosen. The first methods are undoubtedly the most widely used because of their simplicity and general applicability to many different systems. Other methods e.g. size exclusion chromatography, capillary electrophoresis, or spectroscopic methods have been not described. Oravcova et al. (1996) gives a comprehensive review and comparison for these applications. 

Historically, direct procedures were too cumbersome, time-consuming, and expensive for use in a routine clinical laboratory. The introduction of very sensitive immunoassays for T4 and T3 combined with improvements in the dialysis or ultrafiltration of undiluted serum has allowed direct measurement of free thyroid hormones. Thus direct equilibrium dialysis and ultrafiltration methods are available for FT4 measurement. 

FT4 assays based on direct equilibrium dialysis or ultrafiltration measure free hormone without the need for total hormone measurements. These methods are unaffected by either variations in serum binding proteins or thyroid hormone autoantibodies. However, IV heparin administration can cause spurious elevations m FT4 determined by these techniques as a consequence of in vitro generation of free fatty acids. Mean values obtained in euthyroid healthy subjects are reported to be slightly higher when using ultrafiltration methods than when using equilibrium dialysis. Analytical performance goals have been recommended for free thyroid hormone assays.When an FT4 assay... 

Several isotopic methods have been used to accurately estimate the free hormone fraction, including equilibrium tracer dialysis, ultrafiltration, Sephadex chromatography, and polyacrylamide gel filtration. However, these techniques are very cumbersome and time consuming for routine clinical use and, with the exception of equilibrium tracer dialysis, have found little practical application. Unfortunately the free hormone fraction as estimated in normal sera by equilibrium tracer dialysis varies substantially among laboratories. Methodological difficulties include the presence of residual impurities in the tracer, the temperature and duration of dialysis, tlie adsorption of tracer by components of the dialysis system, and the in vitro generation of free fatty acids. ... 

Labeled-Analogue Methods, In a typical one-step RIA for FT4, endogenous free hormone and a I-labeledT4 analogue compete for solid-phase antibody binding sites in the presence of serum protein the amount of labeled analogue bound by the specific antibody is inversely related to the amount of FT, in the specimen. Test results are expressed relative to secondary serum calibrators that have been independently calibrated using equilibrium dialysis or ultrafiltration techniques. A number of one-step RIAs have been developed and are commercially available. 

Reference intervals for free testosterone and percent free testosterone in serum are listed in. Table 53-6 and in Chapter 5 261,336 q jjggg concentrations compare favorably with those obtained when using membrane ultrafiltration and gel filtration methods. Equilibrium dialysis is considered die reference method for determining free testosterone in serum. 

Most problems with this procedure have involved tracer impurities and the separation of bound and free labeled fractions. Several separation techniques have been used, including equilibrium dialysis, membrane ultrafiltration, and steady-state gel filtration. Their deficiencies include a requirement for a large sample volume, the need for complicated correction of sample volume changes that occur during the separation, and difficulties of collecting and measuring radioactivity in numerous fractions of each sample. Equilibrium dialysis has been used most often in the past, but serious errors often arise from the sample dilution required by this method. Symmetrical dialysis of undiluted samples is reported to be less susceptible to tracer contamination and dilution effects. Ultrafiltration also appears to overcome these problems and to obviate errors caused by dilution. 

The classical methods of studying protein binding of drugs include equilibrium dialysis and ultrafiltration. The latter may provide quick measurements but is not necessarily as accurate as the equilibrium dialysis method. Detailed discussions on this may be found in textbooks listed in Suggested Readings. 

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