Kinetic assessment of apparent facilitation by albumin of cellular uptake of unbound ligands

Abstract

Previous studies of the effect of albumin on initial uptake of ligands by isolated cell suspensions or cultures found that the apparent uptake for unbound ligand appeared larger in the presence of binding to the albumin than when albumin was absent. Furthermore, when ligand and albumin were increased in a fixed molar ratio, uptake appeared to be competitively inhibited by the excess albumin. We examined the kinetics underlying this apparent facilitation phenomenon by incorporating unbound fraction of ligand in the medium (fu1) into the general model for diffusion between two compartments. The analysis showed that even in the absence of facilitation by albumin, the apparent rate constant for uptake of unbound ligand (k/fu1) increases as albumin concentration increases but the uptake clearance of unbound ligand remains constant. This theoretical analysis was verified experimentally by measuring the effect of albumin on uptake rates of 14C-taurocholate (12, 24, 48, 60, and 96 microM, with and without 0.87 mM albumin) in a nonphysiological system consisting of two solutions separated by a cellulose membrane. Moreover, when the taurocholate and albumin concentrations were increased in a fixed molar ratio of 0.06 (taurocholate 12-96 microM, albumin 0.2-1.6 mM), the initial uptake rate exhibited the same nonlinear pattern as the previous studies that used living cells. This pattern was due not to saturation of a putative albumin receptor but simply to the concomitant decrease in fu1 which tended to offset the increase in uptake rate due to the increasing total taurocholate concentration. The model was also used to evaluate published data describing the effect of albumin on the uptake of iopanoic acid by cultured hepatocytes. In accordance with the model, k1/fu1 increased as albumin concentration increased, but uptake clearance was independent of albumin concentration. Therefore, the kinetic pattern found in this and other studies with isolated cell suspensions or cultures argues against a special role for albumin in facilitating cellular ligand uptake.

Hepatic pharmacokinetics of taurocholate in the normal and cholestatic rat liver

“…The model also assumed that the uptake and excretion of TC are the main rate determinants for TC transport and that intracellular trafficking by intracellular diffusion of free and bound TC is sufficiently rapid so that intracellular binding and diffusion are not rate limiting. It is possible that the known TC protein binding facilitated transport (Blitzer & Lyons, 1985) may result in apparent 'well-stirred' model behavior as noted by Morgan et al (1990). The poorer fitting of [ 3 H]TC data with both the slow diffusion/ bound and the slow binding models compared to the 'wellstirred' hepatobiliary TC transport model was seen as supportive of the assumption that the intrahepatocellular transport processes were not rate limiting for TC.…”

“…The model also assumed that the uptake and excretion of TC are the main rate determinants for TC transport and that intracellular trafficking by intracellular diffusion of free and bound TC is sufficiently rapid so that intracellular binding and diffusion are not rate limiting. It is possible that the known TC protein binding facilitated transport (Blitzer & Lyons, 1985) may result in apparent ‘well‐stirred’ model behavior as noted by Morgan et al . (1990).…”

The Effect of Protein Binding on the Hepatic First Pass of O-Acyl Salicylate Derivatives in the Rat

“…However, direct anatomical measurements of the size of the unstirred water layer in the intact sinusoid have yet to be determined. Morgan et al(1990) suggested that the kinetic analysis used by others was inappropriate. They used a kinetic model which incorporated the unbound fraction of ligand into the general model for diffusion between two compartments and showed that the apparent rateconstant for unbound ligand concentration increased with albumin concentration, although the uptake clearance of unbound ligand remained constant.…”

Facilitated Diffusion and Membrane Permeation of Fatty Acid in Albumin Solutions

“…There is abundant discussion and debate on this point, nicely reviewed by Weisiger 42 and extended by Schwab and Goresky. 28 Expressing a viewpoint opposite to ours, Morgan et al 21 show that the flux of ligand across an inert membrane between two compartments (mixing chambers without diffusion gradients) was constant over a wide range of total ligand concentrations at constant ligand/albumin ratio, and that the fraction of ligand permeating diminishes as the albumin concentration is increased. Our present model will give these results by setting the diffusion coefficients and k d1 to high values.…”