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Laboratory evaluation of thyroid function
Published in Pallavi Iyer, Herbert Chen, Thyroid and Parathyroid Disorders in Children, 2020
Measuring free hormone concentration: When measuring FT3 or FT4 concentration, one must consider the potential of measuring protein-bound T4 and T3 interacting with the capture antibody and impacting results. To reduce this, one can separate the protein-bound T3 and T4 from the FT3 and FT4 prior to measurement. The two ways to do this are ultrafiltration and equilibrium dialysis. In ultrafiltration, the sample is centrifuged and the ultrafiltrate is then measured (1). In equilibrium dialysis, there is a dialysis membrane which separates the small molecules (FT3 or FT4) from the larger molecules (proteins), allowing for direct measurement of free hormone concentrations (1).
Special Consideration of Drug Disposition
Published in Gary M. Matoren, The Clinical Research Process in the Pharmaceutical Industry, 2020
Other investigators have also concluded that, in the uremic patient, decreased binding of drugs cannot be accounted for on the basis of albumin levels alone. Using equilibrium dialysis to determine protein binding, Craig et al. [26] investigated sulfamethoxazole, dicloxacillin, penicillin G, phenytoin, salicylate, and digitoxin binding in treated and untreated sera from uremic patients and normal subjects. They undertook measurements in serum before and after charcoal treatment at pH 3.0. Treatment of the sera with charcoal did not affect the binding in any way when the serum was from normal subjects. However, charcoal treatment of the serum from uremic subjects led to significantly increased binding in almost every case. Obviously, charcoal treatment had removed one or more substances which either directly or indirectly interfered with protein binding.
Identification Of Receptors In Vitro
Published in William C. Eckelman, Lelio G. Colombetti, Receptor-Binding Radiotracers, 2019
In summary, equilibrium dialysis is best for ligands with relatively low specific activity, when the receptor can be found in high concentration. It is preferable over filter assays when binding has a rather low affinity, especially if the dissociation rate is fast. Equilibrium dialysis is equally effective with soluble and membrane bound receptors. Filter assays are more rapid, and are best for detecting small amounts of receptor using high affinity ligands (Kd below 1 nM) labeled to high specific activity (above 1 Ci/ mmol).
The in vitro metabolism and in vivo pharmacokinetics of the bacterial β-glucuronidase inhibitor UNC10201652
Published in Xenobiotica, 2022
Anna Kerins, Marta Koszyczarek, Caroline Smith, Phil Butler, Rob Riley, Vamsi Madgula, Nilkanth Naik, Matthew R. Redinbo, Ian D. Wilson
In order to measure the extent of binding of UNC10201652 to plasma and microsomal protein, UNC10201652 was added into either plasma or microsomes at a concentration of 2 µM (containing 0.5% DMSO v/v). For these experiments, undiluted pooled mixed gender human, male mouse CD-1 and male rat Sprague Dawley plasma and 0.5 mg/mL human, mouse and rat liver microsomes were used. Buffer (0.1 M phosphate, pH 7.4) was prepared containing 0.5% DMSO v/v to ensure the experiment was maintained at 0.5% final DMSO concentration. The experiment was performed using a Rapid Equilibrium Dialysis (RED) device, with the two compartments separated by a semi permeable membrane. Buffer was added to one side of the membrane and the plasma or microsomal initial solution to the other side. The incubations were performed in triplicate. The RED device and the plasma initial solutions were incubated for 4 h at 37 °C at 5% CO2, with agitation at 128 g to allow the system to reach equilibrium. After equilibration, samples were taken from both sides of the membrane and the plasma initial solutions. Calibration curves were prepared in blank plasma and blank buffer, both containing 0.5% DMSO concentration v/v. The samples and standards were precipitated with 3 volumes of acetonitrile containing internal standard and centrifuged (1280g, 30 min, 4 °C). Supernatant was then diluted with water prior to LC-MS/MS analysis. A control compound was included per matrix in each experiment (amitriptyline for microsomes; warfarin for plasma).
An update on the importance of plasma protein binding in drug discovery and development
Published in Expert Opinion on Drug Discovery, 2021
Low temperature (e.g. 4°C) slows down enzymatic and chemical reactions. It can be used as a generic method to measure binding of unstable compounds. In theory, binding affinity may change when temperature is lowered. However, in practice, low temperature (i.e. 4°C) has been shown to have minimal effects on fu values in plasma and tissues compared to those measured at 37°C using a large set of structurally diverse compounds [29]. Therefore, equilibrium dialysis at low temperature can be used to measure fu of unstable compounds in plasma, tissue homogenate and other matrices. Because of slower diffusion rates of compounds at low temperature, longer incubation time is usually necessary to ensure equilibrium (e.g. 18–24 hours). The advantages of the low temperature binding method is that it is generic and can be used for most of the unstable compounds without knowing the instability mechanisms involved. No method development is needed and, because no inhibitors are used, there are no concerns of inhibitor interference. The low temperature approach has also been applied to measure blood-to-plasma ratio successfully for unstable compounds [58].
Comparison between lab variability and in silico prediction errors for the unbound fraction of drugs in human plasma
Published in Xenobiotica, 2021
Urban Fagerholm, Ola Spjuth, Sven Hellberg
Several methods for determination of fu exist, including equilibrium dialysis, ultrafiltration, ultracentrifugation, turbulent flow chromatography, biosensors, fluorescence spectroscopy, capillary electrophoresis, high-performance liquid chromatography using columns containing immobilised plasma proteins and microdialysis (Cohen 2004). These have their specific advantages and disadvantages. Ultrafiltration and equilibrium dialysis are the most commonly used methods today. Ultrafiltration, which allows automation and rapid determinations, is highly dependent on the extent of non-specific binding of compounds to plastics and the ultrafiltration membrane surface (Kwon 2002; Cohen 2004). The more time consuming equilibrium dialysis is generally accepted as a more accurate method, especially for highly bound compounds (Kwon 2002). However, non-specific binding of compounds to dialysis devices and membranes is also a potential problem with this technique (Kwon 2002). According to Kariv et al. (2001), it is recommended that the equilibrium dialysis assay is not used for compounds with a solubility of less than 5 µM at 37 °C as the insoluble compound will not be able to freely cross the membrane. Another limitation with this technique is the increase of plasma protein concentration in the plasma sample as plasma water is filtrated (Zeitlinger et al. 2011).