Binding of tolbutamide to glycated human serum albumin

Abstract

The presence of elevated levels of glucose in blood during diabetes can lead to the non-enzymatic glycation of serum proteins such as human serum albumin (HSA). This study examined the changes that occur in binding of the sulfonylurea drug tolbutamide to HSA as the level of glycation for this protein was increased. High-performance affinity chromatography was used in this work along with columns containing various preparations of in vitro glycated HSA. It was found in frontal analysis experiments that the binding of tolbutamide with all of the tested preparations of glycated HSA could be described by a two-site model involving both strong and weak affinity interactions. The association equilibrium constants (K(a)) for tolbutamide at its high affinity sites on glycated HSA were in the range of 0.8-1.2 x 10⁵ M⁻¹ and increased by 1.4-fold in going from normal HSA to mildly glycated HSA. It was found through competition studies that tolbutamide was binding at both Sudlow sites I and II on the glycated HSA, in agreement with previous studies. The K(a) for tolbutamide at Sudlow site II increased by 1.1- to 1.4-fold in going from normal HSA to glycated HSA. At Sudlow site I, the K(a) for tolbutamide increased by 1.2- to 1.3-fold in going from normal HSA to the glycated HSA samples. This information demonstrates the effects that glycation can have on drug interactions on HSA and should provide a better quantitative understanding of how the protein binding of tolbutamide in serum may be affected for individuals with diabetes.

Figure 1

Structure of tolbutamide.

Figure 2

(a) Frontal analysis and (b) zonal elution studies for tolbutamide on the gHSA1 column. The tolbutamide concentrations in (a) were (top-to-bottom) 200, 100, 50, 20, and 10 μM. The results in (b) were obtained using R-warfarin as a probe for Sudlow site II, along with tolbutamide concentrations in the mobile phase (top-to-bottom) of 20, 15, 10, 5, and 1 μM.

Figure 3

Double-reciprocal plot of frontal analysis data obtained for tolbutamide on the gHSA1 column. The best-fit line for the upper linear region when analyzed according to Eqn. (5) is y = 670 (± 20) x + [6.1 (± 1.0) × 10⁷], r = 0.999, n = 6. The error bars represent a range of ± 1 SD and are of a comparable size to the data markers in this plot.

Figure 4

Plot of m_Lapp versus [Tolbutamide] for the gHSA1 column, as analyzed according to Eqn. (4) and a two-site model. The best-fit values obtained for the association equilibrium constants and binding capacities are summarized in Table 2. The data used in this plot was the same as in Figure 3. The error bars for the data points (not shown) are comparable in size to the data markers in this plot.

Figure 5

Zonal elution studies on the gHSA1 column for the injection of L-tryptophan as a site-selective probe for Sudlow site II in the presence of tolbutamide as a competing agent. The best-fit line obtained when using Eqn. (6) is y = [1.2 (± 0.1) × 10⁴] x + 0.20 (± 0.01), r = 0.998, n = 5. The error bars represent a range ± 1 SD and are comparable in size to the data markers in this plot.

Figure 6

Zonal elution studies on the gHSA1 column for the injection of R-warfarin as a site-selective probe for Sudlow site I in the presence of tolbutamide as a competing agent. The best-fit line obtained when using Eqn. (6) is y = 1500 (± 100) x + 0.021 (± 0.001), r = 0.999, n = 5. The error bars represent a range of ± 1 SD and are comparable in size to the data markers in this plot.