Hepatocellular sulfobromophthalein uptake at physiologic albumin concentrations: kinetic evidence for a high affinity/low capacity sinusoidal membrane system.
OBJECTIVE
Controversy exists regarding the nature of the hepatocyte membrane transport system for sulfobromophthalein and other organic anions and its driving forces. Most studies have been performed in the absence of albumin, the plasma sulfobromophthalein binding protein, or using very low albumin concentrations. We have shown that in the latter case uptake kinetics reflect dissociation/diffusion events and not membrane transport. In contrast, without albumin very high sulfobromophthalein concentrations reach the cell surface and may overwhelm a high affinity/low capacity system operating in vivo. The aim of this study was to test the latter hypothesis.


METHODS
Sulfobromophthalein uptake was measured by rapid filtration in isolated hepatocytes without albumin (up to 13 microM sulfobromophthalein) and with 600 microM albumin (sulfobromophthalein:albumin from 0.03:1 to 1:1), a physiologic setting which greatly reduces the unbound BSP concentration. Unbound sulfobromophthalein concentration was estimated according to a three binding site model.


RESULTS
In the absence of albumin, kinetic parameters for sulfobromophthalein uptake were similar to those reported in the literature (K(m):7.1 +/- 1.2 microM; V(max): 452 +/- 37 pmol/min/5 x 10(4) cells). In the presence of albumin, sulfobromophthalein uptake displayed much greater affinity and much lower capacity (K(m): 80 +/- 11 nM; V(max): 60 +/- 9 pmol/min/5 x 10(4) cells).


CONCLUSIONS
These findings suggest that in the absence of albumin, resulting high sulfobromophthalein concentrations overload (and make undetectable) a high affinity/low capacity system operating at physiologic albumin concentrations (i.e. at low unbound sulfobromophthalein concentrations). Previously characterized transport systems may be operating only under defined conditions. These findings could explain the apparent controversy regarding the nature of the sulfobromophthalein transport system and its driving forces.
Sorrentino
D
D
Department of Internal Medicine, University of Udine, Italy.
Bartoli
E
E
D000818:Animals; D001667:Binding, Competitive; D007700:Kinetics; D008099:Liver / Q000166:cytology / Q000378:metabolism*; D008297:Male; D009994:Osmolar Concentration; D051381:Rats; D017207:Rats, Sprague-Dawley; D012709:Serum Albumin / Q000378:metabolism*; D013448:Sulfobromophthalein / Q000378:metabolism / Q000493:pharmacokinetics*
D012709:Serum Albumin; D013448:Sulfobromophthalein