CARRIER-MEDIATED UPTAKE AND EXCRETION OF BROMOSULFOPHTHALEIN-GLUTATHIONE IN PERFUSED-RAT-LIVER - A MULTIPLE INDICATOR DILUTION STUDY

The hepatic removal of the glutathione conjugate of bromosulfophthalei n (BSPGSH) was studied in the single-pass perfused rat liver with the multiple indicator dilution (MID) technique against various background concentrations of BSPGSH (20 to 214 mu mol/L) over which nonlinear bi nding to both plasma (albumin) and tissue proteins with two classes of binding sites was found. A bolus containing Cr-51-labeled red blood c ell (a vascular reference), [I-125]albumin and [C-14]sucrose (large an d small molecular weight interstitial references, respectively), D2O ( a cellular space reference), and [H-3]BSPGSH was injected into the por tal vein during steady-state. The eliminated fraction of dose, obtaine d by subtracting the survival fraction of [H-3]BSPGSH in plasma from o ne, corresponded to the steady state extraction ratio (E) with bulk da ta, which declined from 0.74 +/- 0.04 to 0.27 +/- 0.01 with concentrat ion. The major portion of the tracer outflow profile was a throughput component, which is the proportion of tracer that did not enter liver cells during its transit through the liver. The influx, efflux, and se questration coefficients, evaluated with previously developed barrier- limited models, provided the corresponding influx (k(1)), efflux (k(-1 )) and excretion (k(seq)) rate constants. Concentration-dependent infl ux (V-max = 83 nmol min(-1) g(-1) and K-m = 3.7 mu mol/L), efflux (V-m ax = 15 nmol min(-1) g(-1) and K-m = 1.8 mu mol/L), and excretion (V-m ax = 94 nmol min(-1) g(-1) and K-m = 1.8 mu mol/L) were obtained for B SPGSH, when K-m values are expressed in terms of the unbound concentra tions. In these calculations, the observed unbound tissue concentratio n was not used for estimation of the V-max and K-m for efflux and excr etion because of overestimation, because of the presence of highly con centrated BSPGSH in ductular elements present in liver homogenates; ra ther, the unbound tissue concentration was calculated from the influx, efflux, and removal rate coefficients. Because of carrier-mediated en try, the unbound tissue concentration does not equal the unbound plasm a concentration, and kinetic parameters for BSPGSH excretion could be alternately estimated when the rate of excretion or net rate of loss o f BSPGSH from plasma was regressed against the estimated tissue unboun d concentration. This yielded a V-max of 97 nmol min(-1) g(-1) and a K -m of 3.6 mu mol/L, values similar to those obtained from MID. Regress ion of rates of biliary excretion or net plasma disappearance against the logarithmic average unbound concentration, however, yielded a V-ma x of 50 nmol min(-1) g(-1) and a K-m of 3.3 to 3.6 mu mol/L, values wh ich were, as expected, different from those obtained with MID for eith er influx, efflux, or removal. It was found that intracellular BSPGSH levels had not increased sufficiently enough to saturate efflux and ex cretion, and the limiting feature of the transfer process at high-bulk concentration of BSPGSH appeared to be the influx process.