Dialysis involves the separation of two compartments containing differing concentrations of a solute in solution by a semi-permeable membrane. This membrane allows passage of solutes of sufficiently small size from one compartment to the other along a concentration gradient. Theoretically, the solute concentration in both compartments will establish equilibrium such that there is no net flux of solute; the concentration of solute not bound to nonpermeable macromolecules then will be equal in both compartments. The solute diffusion rate, as described by Fick's law, is a function of membrane surface area, thickness, concentration gradient, compartment volume, and ligand diffusion coefficient (43).
Tissue and plasma proteins often bind drugs and other low molecular weight compounds. Hypothesis regarding mechanisms of binding include the generally held view that a reaction occurs between two oppositely charged ions (essentially salt formation). Negatively charged drugs bind to the positively charged amino acid groups, such as histidine or lysine, of plasma proteins. Additional contributions to binding phenomena include hydrophobic interactions (44). Nonpolar functional groups of drug and protein or tissue interact via van der Waals forces.
Pharmacodynamic effects of drugs are considered to be a function of the unbound concentration in plasma (45). For this reason, it is important to determine the unbound (i.e., therapeutically relevant) concentration of pharmacological agents. Dialysis techniques are well suited to make these determinations. In the anterior chamber, low concentrations of proteins are encountered (4). However, under conditions of compromised blood-aqueous barrier, an increased influx of proteins from plasma may result in elevated aqueous protein concentrations (46). Under these conditions, the assessment of unbound concentrations in aqueous humor may become more important in the establishment of the pharmacodynamics arising from intraocular exposures to the substrate in question.
Microdialysis is a dynamic process. Perfusion medium is perfused through the probe. Analyte concentrations in perfusate and in the surrounding medium are not in equilibrium (41). This introduces a number of technical problems that must be overcome in creative ways. Microdialysis is a relatively sophisticated tool. There are a number of challenges to appropriate use of this technique. Although nonspecific binding to the microdialysis membrane is minimized as compared to other dialysis methods, plastic tubing is used to deliver perfusate to the probe and to deliver the dialysate from the probe to the collection vessel. Nonspecific binding to the tubing is possible (47). This situation can be exacerbated when coupling microdialysis directly to other instrumentation since longer tubing usually is required. In experiments examining plasma protein binding of drug in vivo, microdialysis requires sufficient time to achieve stable concentrations. This process requires more time (than ultrafiltration, for example), and recovery of substrate across the membrane can be time- and temperature-dependent.
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