Lactosylation of albumin reduces uptake rate of dibromosulfophthalein in perfused rat liver and dissociation rate from albumin In Vitro

Abstract

Two types of models have recently been proposed to describe hepatic uptake kinetics of protein bound drugs: a model in which dissociation from plasma protein is rate limiting the process, and a model in which an interaction between protein and hepatocyte surface is thought to promote dissociation and uptake of the drug. This study was designed to investigate several aspects of both models, using lactosylated albumin as a binding protein that can interact with the Ashwell receptor abundantly present on the hepatocyte. Dibromosulfophthalein clearance was studied in rat liver in the presence of 150 microM (1%) albumin or 150 microM lactosylated albumin. Initial disappearance rate from perfusate in the presence of lactosylated albumin indicated a 2-fold decrease in hepatic uptake rate compared with native albumin. This was confirmed by compartmental analysis, showing a similar decrease in hepatic uptake rate constant. Protein binding of dibromosulfophthalein to lactosylated albumin was only marginally different from normal albumin. Consequently, modification of the protein retarded uptake of the organic anion at an essentially unchanged unbound concentration. Fluorescence spectroscopy of lactosylated albumin showed a blue-shifted tryptophan emission spectrum compared with albumin, indicating increased hydrophobicity of the neoglycoprotein. We therefore considered a change in off-and-on rate for binding of dibromosulfophthalein in lactosylated albumin. Rapid filtration experiments indicated that the dissociation rate constant of dibromosulfophthalein from lactosylated albumin was half that of controls. We conclude that the decreased off-rate from lactosylated albumin can explain the retarding influence on hepatic uptake rate of dibromosulfophthalein. This observation argues for the concept of dissociation-limited uptake in the hepatic clearance of the organic anion.

An examination of protein binding and protein-facilitated uptake relating to in vitro-in vivo extrapolation

“…To further explore this, van der Sluijs et al (1987) measured dibromosulfophthalein (DBSP) uptake in rat liver perfused with native albumin vs. lactosylated albumin. After demonstrating that DBSP had similar protein binding to the lactosylated albumin as to native albumin, a 40% decrease in the hepatic uptake rate constant for the lactosylated albumin was found.…”

Organic Anion Uptake by Hepatocytes

“…Similar results were found for plasma disappearance of bilirubin in mutant analbuminemic rats (79). Other studies quantified uptake of dibromosulfophthalein (DBSP) bound to albumin or lactosylated albumin by isolated rat liver under recirculating perfusion conditions (177). These studies also showed a reduced off-rate of DBSP from lactosylated as compared to native albumin that corresponded to reduced hepatic uptake.…”

Binding kinetics in drug discovery ndash A current perspective

“…A problem of FI measurements is that both bound and unbound molecules fluoresce, so that their respective contributions to the fluorescence intensity have to be calculated (66) which is prone to errors. Alternatively unbound molecules have to be separated from the complex (117). Further drawbacks result from high background signals due to autofluorescence of cells and cell components as well as the relatively high level of non-specific signals.…”