Macromolecule-Macromolecule Interaction in Drug Distribution. III. Kinetic Characterization of the Uptake of Fractionated [³H]Heparin and the Effect of Plasma Proteins in the Perfused Rat Liver

Abstract

The concentration-dependent hepatic uptake of fractionated [³H]heparin, a macromolecular model drug, was kinetically characterized and the effect of plasma proteins, albumin and α-globulin, was evaluated in the perfused rat liver as part of an ongoing effort to elucidate the mechanism of interaction of macromolecular drugs with biological macromolecules and the role of this interaction in the drugs' distribution. In the absence of proteins, the uptake of fractionated [³H]heparin was saturable with the maximum uptake velocity (V_max) of 7.6 pmol/min/g liver and the Michaelis constant (K_m) of 32.2 nM, suggesting the involvement of a specialized transport. α-Globulin (8.0 mg/ml) reduced the uptake of fractionated [³H]heparin at lower heparin concentrations. However, albumin (40 mg/ml) did not affect the uptake of fractionated [³H]heparin, suggesting an insignificant interaction. Assuming that fractionated [³H]heparin bound to α-globulin cannot be uptaken and that the reduction in uptake was solely attributable to the saturable Scatchard-type binding of fractionated [³H]heparin to α-globulin, the dissociation constant (K_d) and the binding capacity (n) were estimated to be 2.1 nM and 0.002, respectively.In in vitro binding experiments by ultrafiltration, K_d and n were estimated as 168 nM and 0.5, respectively, for α-globulin and 1021 nM and 0.02, respectively, for albumin, suggesting lower affinity and higher capacity in vitro for each protein. The results of in vitro binding experiments did not agree with those of perfusion experiments in that the binding of fractionated [³H]heparin to albumin was significant and the unbound fraction in the presence of α-globulin was smaller than that estimated in the analysis of hepatic uptake. The apparent enhancement of dissociation of fractionated [³H]heparin in perfusion suggests the participation of "protein-mediated transport, " as reported for low molecular weight compounds in the transport of macromolecular compounds.Thus, the present study successfully characterized the hepatic uptake of fractionated [³H]heparin as a Michaelis-Menten type process in the perfused rat liver and confirmed the involvement of protein-mediated transport.