Direct equilibrium dialysis

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. 

Direct Equilibrium Dialysis. In this method, undiluted serum specimens are dialyzed for 16 to 18 hours at 37 °C in a reusable dialysis chamber, " The dialysis buffer provides for minimal changes in the serum matrix. The dialysate is then analyzed directly using a sensitive RIA. The range of reportability is 2 to 128 ng/L (2.6 to 165 pmol/L), and the interassay coefficient of variation is approximately 7%. 

Expected values for FT4 using direct equilibrium dialysis are as follows ... 

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... 

Direct two-step and one-step immunoassays estimate free hormone concentrations by using antibody extraction techniques. Although it is often clauned or implied that these immunoextraction assays measure FT or FT3 directly, virtually all methods do so indirectly by relating test results to extracted serum calibrators with free hormone values that have been independently measured using reference methods (e.g.,. direct equilibrium dialysis/RlA). Further, studies have shown that all of the free hormone estimate methods on current instrument platforms are binding protein dependent to some extent. ... 

Clinical Condition Direct Equilibrium Dialysis/RIA Other Index Methods Two-Step Methods Analogue Methods... 

Estimates of FT4 and FT3 generally give results in healthy subjects, hyperthyroid and hypothyroid patients, and patients with only mild binding protein abnormalities that are comparable with those of reference methods such as direct equilibrium dialysis and RIA assays. In these individ-... 

This assay system developed by Chaires [136] is a new, powerful and effective tool based on the fundamental thermodynamic principle of equilibrium dialysis for the discovery of ligands that bind to nucleic acids with structural and sequence selectivity. Here, identical concentrations of various nucleic acid samples are dialysed in dispodialysers against a common ligand solution. At equilibrium, the contents of the ligand bound to each nucleic acid are determined and this is correlated directly to the ligand s specificity to a particular sequence. 

Valproic acid Direct immersion PDMS (100) GC-FID (LOD l mg/mL) Equilibrium dialysis followed SPME Krogh et al., 1995 (7)... 

It is possible to measure the different single ion interaction coefficients directly by equilibrium dialysis (Bai et al., 2007 Strauss et al., 1967). An alternative method for measuring T2+ takes advantage of a fluorescent dye that chelates Mg2+ (Grilley et al., 2006). In essence, the method uses the dye to sense differences in the Mg2+ activity in the presence or absence of an RNA. Detailed theoretical justification, titration protocols, and data analysis for the method have been presented elsewhere (Grilley et al., 2009). For the A-riboswitch, titrations carried out in the presence or absence of ligand measure T2+ for the folded or unfolded state of the RNA, respectively... 

Nelson J, Tomei R. Direct determination of free thyroxin in undiluted serum by equilibrium dialysis/radioimmunoassay. Clin Chem 1988 34 1737-44. 

Although complex, these calculations have demonstrated a very good correlation (both linearity and value agreement) with equilibrium dialysis measurements and are considered a reliable indicator of free testosterone. The reader is directed to other references for further details on this method, Conditions resulting in abnormal plasma protein concentrations, such as nephrotic syndrome, cirrhosis, and pregnancy require adjustments in the assumption for albumin concentration. 

Once the structure of the PBPK model is formulated, the next step is specifying the model parameters. These can be classified into a chemical-independent set of parameters (such as physiological characteristics, tissue volumes, and blood flow rates) and a chemical-specific set (such as blood/tissue partition coefficients, and metabolic biotransformation parameters). Values for the chemical-independent parameters are usually obtained from the scientific literature and databases of physiological parameters. Specification of chemical-specific parameter values is generally more challenging. Values for one or more chemical-specific parameters may also be available in the literature and databases of biochemical and metabolic data. Values for parameters that are not expected to have substantial interspecies differences (e.g., tissue/blood partition coefficients) can be imputed based on parameter values in animals. Parameter values can also be estimated by conducting in vitro experiments with human tissue. Partitioning of a chemical between tissues can be obtained by vial equilibration or equilibrium dialysis studies, and metabolic parameters can be estimated from in vitro metabolic systems such as microsomal and isolated hepatocyte syterns. Parameters not available from the aforementioned sources can be estimated directly from in vivo data, as discussed in Section 43.4.5. 

Equilibrium dialysis methods often involve the use of radioactively labelled ligands, so that very small concentrations and very tight binding can be measured directly. 

Equilibrium dialysis methods can be used to measure binding interactions directly. 

The slope of the double-reciprocal plot" (I/[PL] versus 1/[L]) = ]/KCf. This is useful because only equilibrium dialysis (and related methods) gives free the ligand concentration [L] directly. For most other methods we need to make approximations, or lit to complete binding expression. 

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