Equilibrium dialysis protein binding assays

FIGURE 6.6 Experimental layout for (A) permeability assays using PAMPA, (B) permeability assays using Caco-2, and (C) equilibrium dialysis protein binding assays. 

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-... 

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. ... 

If CLint is determined in vitro and if / is known, the important PK parameters (CLh, F, and h) ean be directly estimated using Eq. 13.14-13.16, assuming that Qh is constant, that is, 1.5 L/min, or approximately 21 mL/min/ kg for humans (Davies and Morris, 1993). Moreover, if nonspecific protein binding in the in vitro assay systems is factored into the equations, / should be replaced by //u, where/ is the unbound fraction in the in vitro metabolic systems, whieh can be determined using equilibrium dialysis (Soars et al., 2002). Therefore, Eq. 13.14 can be expressed as... 

As expected, the metabolism of 5-fluorouraciI, the first metabolite of 5 -deoxy-5-fluorouridine, turned out to be similar [21]. Interestingly, the intermediate, a-fiuoro-P-guanidinopropanoic acid, could not be detected in this study, which utilized F NMR and FIPLC methods. Martino et al. also developed a F NMR assay method to determine the extent of protein binding of 5 -deoxy-5-fluorouridine, which was as valid as equilibrium dialysis [22]. 

Survival of the Mn-binding site(s) in the CaClo-treated PS n membranes was assayed as described (2). Binding of light-harvesting Chi a/b protein (LHC) to Mn was examined by an equilibrium dialysis. LHCs were isolated from CaCl2-treated PS n membranes by centrifugation (224,000 x g, 4 hr) in the presence of 60 mM octylglucoside. After the... 

Property Structural integrity Solubility (nephelometry) Stability (buffer, plasma) Serum protein binding (see Section 6.3.2.4) Assessing physicochemical properties Providing context to in vivo data interpretation Assay (protein binding) Serum from multiple species 5-10 pM compound concentration Ultrafiltration or equilibrium dialysis in 96-well format Analysis Samples in both buffer and serum matrices " PPT followed by LC-MS/MS... 

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