Carrier-mediated uptake and excretion of bromosulfophthalein-glutathione in perfused rat liver: A multiple indicator dilution study

Abstract

The hepatic removal of the glutathione conjugate of bromosulfophthalein (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 mumol/L) over which nonlinear binding to both plasma (albumin) and tissue proteins with two classes of binding sites was found. A bolus containing 51Cr-labeled red blood cell (a vascular reference), [125I]albumin and [14C]sucrose (large and small molecular weight interstitial references, respectively), D2O (a cellular space reference), and [3H]BSPGSH was injected into the portal vein during steady-state. The eliminated fraction of dose, obtained by subtracting the survival fraction of [3H]BSPGSH in plasma from one, corresponded to the steady state extraction ratio (E) with bulk data, which declined from 0.74 +/- 0.04 to 0.27 +/- 0.01 with concentration. 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 sequestration coefficients, evaluated with previously developed barrier-limited models, provided the corresponding influx (k1), efflux (k-1) and excretion (kseq) rate constants. Concentration-dependent influx (Vmax = 83 nmol min-1 g-1 and Km = 3.7 mumol/L), efflux (Vmax = 15 nmol min-1 g-1 and Km = 1.8 mumol/L), and excretion (Vmax = 94 nmol min-1 g-1 and Km = 1.8 mumol/L) were obtained for BSPGSH, when Km values are expressed in terms of the unbound concentrations. In these calculations, the observed unbound tissue concentration was not used for estimation of the Vmax and Km for efflux and excretion because of overestimation, because of the presence of highly concentrated BSPGSH in ductular elements present in liver homogenates; rather, the unbound tissue concentration was calculated from the influx, efflux, and removal rate coefficients. Because of carrier-mediated entry, the unbound tissue concentration does not equal the unbound plasma concentration, and kinetic parameters for BSPGSH excretion could be alternately estimated when the rate of excretion or net rate of loss of BSPGSH from plasma was regressed against the estimated tissue unbound concentration. This yielded a Vmax of 97 nmol min-1 g-1 and a Km of 3.6 mumol/L, values similar to those obtained from MID.(ABSTRACT TRUNCATED AT 400 WORDS)

Transport Is Not Rate-Limiting in Morphine Glucuronidation in the Single-Pass Perfused Rat Liver Preparation

“…Before surgery, animals were anesthetized with an intraperitoneal injection of 50 mg/kg sodium phenobarbital. The surgical procedure and perfusion apparatus were identical to those described previously (Geng et al, 1995). Perfusate consisted of 20% washed fresh bovine RBCs (a kind gift of Ryding Regency Meat Packers Ltd., Toronto, ON, Canada), 4% BSA, 5 mM glucose, and a complement of 20 amino acids in Krebs-Henseleit bicarbonate solution buffered to pH 7.4 and was oxygenated with carbogen (95% oxygen and 5% carbon dioxide; Matheson, Mississauga, ON, Canada).…”

“…Only one concentration was used for each perfusion study. An O (0.096 Ϯ 0.013 ml) was introduced into the inflow system by an electronically controlled HPLC injection valve at 30 min, as described previously (Geng et al, 1995). Outflow samples were rapidly collected immediately at successive 1-(35 samples), 2-(35 samples), and 3-s (20 samples) intervals.…”

Summer Research Program - 1998 Summer Research Extension Program Volume 1 Armstrong Laboratory

“…R6: Previous studies have determined a maximum uptake rate for BSP-GSH of 50 ^mole/hr (for a 10 g liver) (Geng et al, 1995). Since this is so close to the 60 (imole/hr value used for BSP uptake (R2), the decision was made to make both UAmax values equal to 60 nmole/hr.…”

“…Its value was iteratively determined. The presence of a second low affinity transport system for BSP transport has been documented before (Sorrentino et al, 1994;Schwenk et al, 1976).R6: Previous studies have determined a maximum uptake rate for BSP-GSH of 50 ^mole/hr (for a 10 g liver) (Geng et al, 1995). Since this is so close to the 60 (imole/hr value used for BSP uptake (R2), the decision was made to make both UAmax values equal to 60 nmole/hr.…”

“…These parameters were based on earlier studies (Baker and Bradley, 1966). R5: Previous reports indicate that the dissociation constant K<i (= kofi/km) of BSP-GSH is 2 orders of magnitude larger than for BSP (Baker and Bradley, 1966;Geng et al, 1995). Therefore, koff was set to a value 100 times larger than for BSP.…”