Binding affinity and capacity for the uremic toxin indoxyl sulfate

Abstract

Protein binding prevents uremic toxins from removal by conventional extracorporeal therapies leading to accumulation in maintenance dialysis patients. Weakening of the protein binding may enhance the dialytic elimination of these toxins. In ultrafiltration and equilibrium dialysis experiments, different measures to modify the plasma binding affinity and capacity were tested: (i), increasing the sodium chloride (NaCl) concentration to achieve a higher ionic strength; (ii), increasing the temperature; and (iii), dilution. The effects on the dissociation constant K(D) and the protein bound fraction of the prototypical uremic toxin indoxyl sulfate (IS) in plasma of healthy and uremic individuals were studied. Binding of IS corresponded to one site binding in normal plasma. K(D) increased linearly with the NaCl concentration between 0.15 (K(D) = 13.2 ± 3.7 µM) and 0.75 M (K(D) = 56.2 ± 2.0 µM). Plasma dilution further reduced the protein bound toxin fraction by lowering the protein binding capacity of the plasma. Higher temperatures also decreased the protein bound fraction of IS in human plasma. Increasing the NaCl concentration was effective to weaken the binding of IS also in uremic plasma: the protein bound fraction decreased from 89% ± 3% to 81% ± 3% at 0.15 and 0.75 M NaCl, respectively. Dilution and increasing the ionic strength and temperature enhance the free fraction of IS allowing better removal of the substance during dialysis. Applied during clinical dialysis, this may have beneficial effects on the long-term outcome of maintenance dialysis patients.

Figures

Figure 1

Scatchard plot of the protein binding of indoxyl sulfate (A–C) and corresponding binding curves (D–F) in normal human plasma. Plasma was 1:2 diluted and incubated at 0.15 M NaCl (A,D) and 0.75 M NaCl (B,E). 1:10-diluted plasma was incubated only at 0.15 M NaCl (C,F). Each experiment was performed with plasma from three different donors (squares: donor 1, triangles: donor 2, crosses: donor 3, and circles: donor 4). Indoxyl sulfate bound to one high affinity binding site in normal plasma. Regression coefficients r2 were: (A) 0.95, 0.98, and 0.96; (B) 0.96, 0.89, and 0.97 for donors 1, 2, and 3, respectively; (C) 0.94, 0.92 and 0.75 for donors 1, 3 and 4, respectively. Regression coefficient r2 of the binding curves; (D–F) was always 1.00.

Figure 2

Effect of the ionic strength on the dissociation constant KD (solid line) and the ratio KD/Bm (dotted line) for indoxyl sulfate in 1:2-diluted plasma. KD and KD/Bm correlate with the ionic strength according to KD = 71.2 × NaCl concentration + 5.1 and KD/Bm = 0.20 × NaCl concentration + 0.03, respectively. *p < 0.05 versus 0.15 M NaCl.

Figure 3

Effect of an increased NaCl concentration on the protein bound fraction of indoxyl sulfate in uremic (A); n = 15, free and bound native toxin concentrations) and healthy (B); n = 18 (3 different donors spiked with 6 different toxin concentrations), free and bound toxin concentrations) human plasma. Increasing ionic strength led to lower protein bound fractions of indoxyl sulfate in both uremic and normal plasma. *p < 0.05.

Figure 4

Theoretical course of the protein bound fraction as a function of KD/Bm for different toxin-protein ratios α according to Equation (1). α and KD/Bm were varied from 0.01 to 1 mol/mol and from 10−4 to 102 mol/mol, respectively. The binding curve corresponds to a single high-affinity binding site on protein.