On the design and interpretation of experiments to elucidate albumin-dependent hepatic uptake.

The liver's apparently anomalous extraction of organic anions tightly bound to albumin continues to provoke controversy and confusion. Decisive experiments have proved difficult to design, and mathematical models have usually been constructed to defend one or another putative mechanism to the exclusion of others. To stimulate more decisive experiments and as an aid to interpreting those already reported, we discuss a general formulation of the problem that predicts the clearance pattern to be expected when facilitated dissociation and codiffusion are joint determinants of the uptake flux.

Beta-lactoglobulin enhances the uptake of free palmitate by hepatocyte monolayers: the relative importance of diffusion and facilitated dissociation.

We compared the uptake of bound palmitate by rat hepatocytes to its uptake by polyethylene using beta-lactoglobulin (BLG) as the binding protein. The experiments were designed to supply a direct measure of the protein-dependent change in the diffusive conductance of extracellular fluid without determining the diffusion coefficients for free and bound fatty acid or the off-rate constant for protein binding. Rate-limiting dissociation in the stirred phase of extracellular fluid was excluded.

Palmitate uptake by cultured hepatocytes: albumin binding and stagnant layer phenomena.

We compared uptake of palmitate by hepatocyte monolayers with uptake by polyethylene membranes under conditions of identical binding and stirring. Hepatocytes and polyethylene display similar clearances when the fatty acid is free, reflecting partial rate limitation by diffusion across the unstirred water layer. When palmitate is 99.

Protein binding of palmitate measured by transmembrane diffusion through polyethylene.

Thin polyethylene membranes permit ready diffusion of protonated long-chain fatty acids but are impermeable to protein and ions. This circumstance recommends polyethylene for measuring the free fraction of fatty acids in the presence of a binding protein and for estimating the ionization constant with which to compute the equilibrium constant for the binding of fatty acid anions. As an example of this approach we report the binding of tracer palmitate to bovine albumin and bovine beta-lactoglobulin.

Only free bile acid drives ileal absorption of taurocholate.

We compared the intestinal absorption of monomeric taurocholate bound to albumin with the absorption of the free form. In the presence of albumin the apparent uptake coefficient is about three times greater than that in its absence. This result resembles the one reported previously for the liver.

Palmitate uptake by hepatocyte monolayers. Effect of albumin binding.

The uptake of 14C-palmitate by rat liver cell monolayers is depressed by binding of the fatty acid to albumin. When the uptake flux is divided by the concentration of free palmitate in the bathing medium, however, the resulting clearance is approximately 14 times greater in the presence of albumin than in its absence. These findings are not accounted for by the different diffusion rates of free and bound palmitate across an unstirred fluid layer, nor attributable to nonequilibrium binding.

Effects of unstirred Disse fluid, nonequilibrium binding, and surface-mediated dissociation on hepatic removal of albumin-bound organic anions.

Proceeding from the observation that organic anions bound to albumin have hepatic extraction fractions that are unexpectedly high, we have studied a distributed model that accounts for this phenomenon by invoking sites on the cell surface that catalyze the dissociation of albumin-anion complexes. The present report extends this model to include nonequilibrium binding and rate-limiting diffusion of bound anion to the cell surface. Simulation analysis of the extended model provides an unambiguous basis for interpreting the apparent intrinsic clearance of free anion.

Hepatic transport and binding of rose bengal in the presence of albumin and gamma globulin.

Gamma globulin and albumin are compared with respect to their effects on the hepatic transport of rose bengal and with respect to the rates and affinities with which they bind this dye. The apparent intrinsic clearance of rose bengal is greater in the presence of albumin than in the presence of gamma globulin, and this difference increases with the protein concentration. Because the binding affinities of these proteins also differ, however, it cannot be concluded decisively that the mechanisms of dye removal are distinct.

Effect of a transported ligand on the binding of albumin to rat liver cells.

Organic anions destined for hepatic uptake often bind to albumin in the circulation. Because albumin binds to liver cells but is not transported, we suggest that sites on the hepatocyte surface catalyze the dissociation of albumin-anion complexes, thus making more free anion available for transport than would otherwise occur. To learn whether liver cells distinguish between free albumin and albumin-anion complexes, we measured the binding of 125I-albumin to isolated rat hepatocytes in the presence and absence of rose bengal, a transported anion that binds extensively to albumin.

Determining hepatic transport kinetics by mathematical modeling.

Three approaches to the measurement of hepatic transport kinetics by mathematical modeling are briefly reviewed. The so-called lumped, two-compartment model and the single-pass multiple-indicator dilution method enjoy a long history of wide application, but each is subject to important errors inherent in their underlying assumptions. An understanding of the reasons for these errors, as revealed by simulation analysis, suggests a third approach that can substantially improve the previous difficulties.

Albumin-mediated transport of rose bengal by perfused rat liver. Kinetics of the reaction at the cell surface.

Rapid dissociation of organic anions from plasma albumin maximizes the presentation of free ligand to the cell surface and thus favors its efficient hepatic extraction. Even assuming these optimal conditions, however, taurocholate and rose bengal have hepatic extraction fractions that are higher than can be accounted for by spontaneous dissociation of their albumin-ligand complexes. In this study we developed a transport model that attributes this behavior to sites on the hepatocyte plasma membrane that bind the albumin-ligand complexes, promoting the transport of ligand into the hepatocyte.

Analyzing tracer disappearance curves to study hepatic transport kinetics.

This paper is devoted to a discussion of recent developments in the compartmental analysis of hepatic transport, especially the interpretation of tracer disappearance curves recorded from the reservoir of an isolated perfused liver preparation. The emphasis is on the advantages that this approach enjoys over other mathematical models, and a critical review of alternative methods is therefore included. The mathematical equations are largely suppressed, however, having appeared elsewhere in detail.

Hepatic transport kinetics: effect of anatomic and metabolic heterogeneity on estimates of the average transfer coefficients.

In the accompanying paper we explore the analysis of solute disappearance curves using conventional linear assumptions. In this study we extend the analysis to examine the possibility of using the same model to recover the average kinetic parameters when the liver lobule displays anatomic and metabolic heterogeneity. The results show that the linear model can be used to recover good estimates of the average transfer coefficients, even though the kinetic parameters that govern uptake, efflux, and excretion are nonlinear functions of sinusoidal length.

How to measure first-order hepatic transfer coefficients by distributed modeling of a recirculating rat liver perfusion system.

A digital computer is used to simulate solute disappearance curves evolving in the reservoir of a rat liver perfusion system. The results show that it is feasible to obtain good estimates of these kinetic parameters with only approximate information about the distribution of sinusoidal flows and the transfer function of the nonexchanging vasculature. By contrast satisfactory estimates of the transfer coefficients for uptake and efflux (but not excretion) are critically dependent on accurate measurements of the system transit times.

Effect of albumin binding on the hepatic transport of rose bengal: surface-mediated dissociation of limited capacity.

We have studied the steady-state removal of rose bengal by perfused rat livers to decide whether the tight association of this anion with albumin is consistent with the conventional view that only the free fraction in the sinusoidal lumen is available for hepatic transport. The results show that the concentration of free dye is not nearly high enough to account for the observed removal rate. From these observations we argue that the liver cell surface reduces the affinity of rose bengal for albumin by a factor of approximately 100 below that observed in free solution.

Simulation and analysis of hepatic indicator dilution curves.

The "multiple-indicator dilution method" of measuring hepatic transport kinetics is subjected to simulation analysis. The objective is to examine the errors that may arise from treating the nonexchanging vasculature as a simple delay and to study the information content of simulated venous outflow curves. We find that the method cannot be counted on to provide consistently reliable estimates of either the transport rate constants or the sinusoidal volume.