Single-pass Perfused Rat Liver Research Articles

Hepatic interaction between quinidine and digoxin: Role of inhibition of sinusoidal Na+/K+ ATPase digoxin binding

The mechanism by which quinidine affects hepatic digoxin pharmacokinetics remains controversial. Here, we study the role of displacement of digoxin from hepatic sinusoidal binding sites by quinidine. We used the impulse–response technique in the single-pass perfused rat liver to describe the digoxin hepatic disposition by a physiologically-based pharmacokinetic liver model. The impulse–response study involved analysis of outflow curves following two consecutive doses of digoxin (42 and 125μg) without and with quinidine (10μM) in perfusate. In addition, the effect of quinidine on digoxin binding in liver subcellular fractions was quantified. Quinidine increased the peak outflow concentration for digoxin at the low digoxin dose but not at the high dose. This increase could be adequately described when digoxin displacement from sinusoidal and intrahepatic binding sites was included in the model. Inhibition of digoxin binding by quinidine was also observed in vitro. The decrease of biliary excretion of digoxin by quinidine was accompanied by a linear increase in sinusoidal efflux of digoxin’s primary metabolite, digoxigenin bisdigitoxoside (Dg2). In contrast to biliary excretion, inhibition of sinusoidal uptake may become dominant only for high concentrations of quinidine.

Nitric oxide induced sinusoidal relaxation after a propranolol priming dose in the perfused rat liver reduces propranolol availability on subsequent dosing

1. The present study sought to explain the mechanism leading to reduced availability of propranolol when given after a priming dose in the single-pass perfused rat liver. 2. Extracellular sucrose space (as a measure of sinusoidal relaxation) in perfused rat liver before and after propranolol or propranolol and N(G)-nitro-L-arginine methyl ester (L-NAME; nitric oxide (NO) synthase inhibitor) treatment were examined. The results showed that propranolol induces sinusoidal relaxation in the perfused liver and this effect could be abolished by NO synthase inhibitor L-NAME. 3. Two bolus injections of propranolol were given to the isolated perfused rat liver and outflow concentration-time profiles of intact propranolol were determined. A two-phase physiologically based organ pharmacokinetic model was applied to estimate hepatocellular influx, efflux, binding, ion-trapping and metabolic elimination pharmacokinetic parameters for propranolol. The recovery of propranolol in the second injection was approximately 54% of that in the first injection. The permeability-surface area product, the binding and the intrinsic clearance all increased significantly after prior exposure of the rat liver to the first bolus of propranolol (P < 0.05). 4. Based on the findings of the present study, we propose that the most likely explanation for the reduced availability of a second propranolol dose (after administration of a priming dose) in the perfused liver is a consequence of the NO-mediated sinusoidal relaxation effect of propranolol, arising from the priming dose. This observation supports the view that the pharmacokinetics of some drugs might be altered by the pharmacodynamic effects of the same drug given earlier in the perfused liver.

Physiologically Based Synthetic Models of Hepatic Disposition

Current physiologically based pharmacokinetic (PBPK) models are inductive. We present an additional, different approach that is based on the synthetic rather than the inductive approach to modeling and simulation. It relies on object-oriented programming. A model of the referent system in its experimental context is synthesized by assembling objects that represent components such as molecules, cells, aspects of tissue architecture, catheters, etc. The single pass perfused rat liver has been well described in evaluating hepatic drug pharmacokinetics (PK) and is the system on which we focus. In silico experiments begin with administration of objects representing actual compounds. Data are collected in a manner analogous to that in the referent PK experiments. The synthetic modeling method allows for recognition and representation of discrete event and discrete time processes, as well as heterogeneity in organization, function, and spatial effects. An application is developed for sucrose and antipyrine, administered separately and together. PBPK modeling has made extensive progress in characterizing abstracted PK properties but this has also been its limitation. Now, other important questions and possible extensions emerge. How are these PK properties and the observed behaviors generated? The inherent heuristic limitations of traditional models have hindered getting meaningful, detailed answers to such questions. Synthetic models of the type described here are specifically intended to help answer such questions. Analogous to wet-lab experimental models, they retain their applicability even when broken apart into sub-components. Having and applying this new class of models along with traditional PK modeling methods is expected to increase the productivity of pharmaceutical research at all levels that make use of modeling and simulation.

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

Binding, transport, and metabolism are factors that influence morphine (M) removal in the rat liver. For M and the morphine 3beta-glucuronide metabolite (M3G), modest binding existed with 4% bovine serum albumin (unbound fractions of 0.89 +/- 0.07 and 0.98 +/- 0.09, respectively), and there was partitioning of M into red blood cells. Transport studies of M (<750 microM) showed similar, concentration-independent uptake clearances (CLs) of 1.5 ml min(-1) g(-1) among zonal and homogeneous, isolated rat hepatocytes. Transport of M3G, ascertained in multiple indicator dilution studies at various steady-state M3G concentrations (10-262 microM), uncovered a low and concentration-independent influx clearance (<10% of flow rate). The outflow dilution curve of [(3)H]M3G was superimposable onto that of [(14)C]sucrose, the extracellular reference, displaying similarity in transit times (23.5 and 22.2 s), negligible biliary excretion, and almost complete dose recovery from perfusate. In contrast, M3G occurred abundantly in both perfusate and bile in single-pass perfusion studies of the precursor, M, and revealed a biliary clearance of formed M3G that was 12.3-fold that of preformed M3G, suggesting a sinusoidal, diffusional barrier for M3G. With increasing concentrations of M (9-474 microM), clearance decreased, and metabolism and biliary excretion displayed concentration-dependent kinetics. Fitting of the data to a physiologically based liver model revealed that M removal mechanisms were saturable, with a K(m,met) of 52.2 microM and V(max,met) of 58.8 nmol min(-1) g(-1) for metabolism, and a K(m,ex) of 41.2 microM and V(max,ex) of 8.1 nmol min(-1) g(-1) for excretion. Sinusoidal transport was not rate-limiting for M removal.

Uptake of l-Carnitine and Its Short-Chain Ester Propionyl-l-carnitine in the Isolated Perfused Rat Liver

Hepatic uptake of propionyl-L-carnitine (PLC) and L-carnitine (LC) was assessed with the impulse-response technique in the single-pass perfused rat liver. The experiments involved a rapid injection (impulse) of a mixture of the radiolabeled test compound (PLC or LC) and a reference compound (sucrose) into portal vein inflow and collection and radiochemical analysis (response) of the venous outflowing perfusate samples. The impulse injection was made in the presence of increasing unlabeled background concentrations of PLC (0-50 microM) or LC (50-500 microM) perfusing the liver. The hepatic uptake was minimal or negligible for LC, whereas the hepatic influx clearance was found to be low (0.095 ml/s equivalent to 5.7 ml/min) for PLC relative to the perfusate flow rate (30 ml/min). When background concentrations of PLC were increased (from 1-50 microM), the influx clearance was reduced in a concentration-dependent behavior, indicating partial saturation of the entry of compound into hepatocytes. PLC was taken up into hepatocytes via a unidirectional transport process with negligible efflux. The hepatic uptake of PLC was significantly reduced in the presence of unlabeled LC (500 microM), indicating an inhibition of the sinusoidal membrane transport of PLC by LC. The study showed the sinusoidal membrane is a permeability barrier to the entry of PLC and LC into hepatocytes, and it is the site of a common carrier-mediated transporter for both compounds.

Moment analysis of metabolic heterogeneity: conjugation of benzoate with glycine in rat liver studied by multiple indicator dilution technique.

Metabolic zonation was assessed with the multiple indicator dilution (MID) technique in the single-pass perfused rat liver with use of moment analysis of the formed metabolite (M) data. During single-pass, retrograde rat liver perfusion with 17 Awake M benzoate, a bolus containing tracer preformed metabolite (PM) [(3)H]hippurate was injected rapidly into the hepatic vein at 20 min postperfusion, followed by injection of a second bolus containing [(14)C]benzoate at 30 min. Both doses also contained noneliminated reference indicators ((51)Cr-labeled RBCs, (125)I-labeled albumin, [(14)C]- or [(3)H]sucrose, and (2)H(2)O). The steady-state extraction ratio of benzoate, the area under the curve (AUC) and its mean transit time (MTT) during retrograde flow were identical to those previously observed for prograde flow. Values of AUC(PM) and MTT(PM) and AUC(M) were also similar to previously published prograde data, but the MTT(M) with retrograde perfusion was smaller than that for prograde perfusion. This, according to theory based on the tubes-in-series model, was consistent with perivenous enrichment of glycination activity when transport of drug was even and when the ratio of drug influx/efflux coefficient exceeded that for metabolite. Similar benzoate transport in periportal, homogeneous and perivenous isolated rat hepatocytes existed, and the influx/efflux coefficients (partition ratio) of benzoate from MID indeed exceeded that of hippurate. However, metabolism by zonal hepatocytes failed to reveal the anticipated metabolic zonation, and this is likely due to the shallow gradient of metabolic activity. The study demonstrates that moment theory is useful in delineating the perivenous enrichment of glycine conjugation activity.

Transport of fluorescent bile acids by the isolated perfused rat liver: kinetics, sequestration, and mobilization.

Hepatocyte transport of six fluorescent bile acids containing nitrobenzoxadiazolyl (NBD) or a fluorescein derivative on the side chain was compared with that of natural bile acids using the single-pass perfused rat liver. Compounds were infused at 40 nmol/g liver min for 15 minutes; hepatic uptake and biliary recovery were measured; fractional extraction, intrinsic basolateral clearance, and sequestration (nonrecovery after 45 minutes of additional perfusion) were calculated. Fluorescent bile acids were efficiently extracted during the first 3 minutes (70%-97%), but net extraction decreased with time mostly because of regurgitation into the perfusate. For cholylglycine and ursodeoxycholylglycine (UDC-glycine), extraction was 94% to 99%, and regurgitation did not occur. Intrinsic hepatic clearance of fluorescent bile acids (2-7 mL/g liver x min) was lower than that of cholylglycine (9.0 +/- 0.6; mean +/- SD) and UDC-glycine (21.4 +/- 0.4). Sequestration at 60 minutes was 8% to 26% for fluorescent bile acids with a cholyl moiety (cholylglycylaminofluorescein [CGamF], cholyllysylfluorescein [C-L-F], cholyl-[N epsilon-NBD]-lysine [C-L-NBD], and cholylaminofluorescein [CamF]), 32% for ursodeoxycholylaminofluorescein (UDCamF), and 88% for ursodeoxycholyl-(N epsilon-NBD)lysine (UDC-L-NBD). Cholylglycine and UDC-glycine had <3% retention. Biliary secretion of sequestered UDCamF, but not of UDC-L-NBD, was induced by adding dibutyryl cyclic adenosine monophosphate (DBcAMP) to the perfusate, possibly by translocation to the canaliculus of pericanalicular vesicles containing fluorescent bile acids. Biliary secretion of UDC-L-NBD, but not of UDCamF, was induced by adding cholyltaurine or UDC-taurine, possibly by inhibition of binding to intracellular constituents or of transport into organelles. It is concluded that fluorescent bile acids are efficiently transported across the basolateral membrane, but in contrast to natural conjugated bile acids, are sequestered in the hepatocyte (UDC derivatives > cholyl derivatives). Two modes of hepatic sequestration of fluorescent bile acids were identified. Fluorescent bile acids may be useful to characterize sequestration processes during bile acid transport through the hepatocyte.

Hepatic uptake of hippurate: a multiple-indicator dilution, perfused rat liver study.

The hepatic transport of hippuric acid (HA), a glycine-conjugated metabolite of benzoic acid that exhibits only modest plasma albumin binding (binding association constant of 2.1 x 10(3) M-1), was studied in the single-pass perfused rat liver (12 ml/min), using the multiple indicator dilution (MID) technique. The venous recovery of [3H]HA on portal venous injection of a MID dose containing a mixture of a set of noneliminated reference indicators and [3H]HA revealed a survival fraction of unity, corroborating the lack of disappearance of bulk HA from plasma. When the outflow recovery was fitted to the barrier-limited model of Goresky et al. (C.A. Goresky, G. G. Bach, and B. E. Nadeau. J. Clin. Invest. 52: 991-1009, 1973), the derived influx (P(in)S) and efflux (P(out)S) permeability-surface area products were found to be dependent on the concentration of HA (1-930 microM); P(in)S and P(out)S were approximately 3.5 times the plasma flow rate at low HA concentration, but decreased with increasing HA concentration. All values, however, greatly exceeded the expected contribution from passive diffusion, because the equilibrium distribution ratio of chloroform to buffer for HA was extremely low (0.0001 at pH 7.4). The tissue equilibrium partition coefficient (P(in)/P(out), or ratio of influx to efflux rate constants, k1/k-1) was less than unity and decreased with concentration. The optimized apparent Michaelis-Menten constant and maximal velocity were 182 +/- 60 microM and 12 +/- 4 nmol.s-1.g-1, respectively, for influx and 390 +/- 190 microM and 29 +/- 13 nmol.s-1.g-1, respectively, for efflux. In the presence of L-lactate (20 mM), however, P(in)S for the uptake of HA (174 +/- 3 microM) was reduced. Benzoic acid (10-873 microM) was also effective in reducing hepatic uptake of HA (5.3 +/- 0.9 microM). These interactions suggest that MCT2, the monocarboxylate transporter that mediates the hepatic uptake of lactate and other monocarboxylic acids, may be involved in HA transport.

Application of the dispersion model for description of the outflow dilution profiles of noneliminated reference indicators in rat liver perfusion studies.

The dispersion model (DM) is a stochastic model describing the distribution of blood-borne substances within organ vascular beds. It is based on assumptions of concurrent convective and random-walk (pseudodiffusive) movements in the direction of flow, and is characterized by the mean transit time (t) and the dispersion number (inverse Peclet number), DN. The model is used with either closed (reflective) boundary conditions at the inflow and the outflow point (Danckwerts conditions) or a closed condition at the inflow and an open (transparent) condition at the outflow (mixed conditions). The appropriateness of DM was assessed with outflow data from single-pass perfused rat liver multiple indicator dilution (MID) experiments, with varying lengths of the inflow and outflow catheters. The studies were performed by injection, of bolus doses of 51Crlabeled red blood cells (vascular indicator), 125I-labeled albumin and [14C] sucrose (interstitual indicators), and [3H]2O (whole tissue indicator) into the portal vein at a perfusion rate of 12 ml/ min. The outflow profiles based on the DM were convolved with the transport function of the catheters, then fitted to the data. A fairly good fit was obtained for most of the MID curve, with the exception of the late-in-time data (prolonged tail) beyond 3 x [symbol: see text]. The fitted DNS were found to differ among the indicators, and not with the length of the inflow and outflow catheters. But the differences disappeared when a delay parameter, t0 = 4.1 +/- 0.7 sec (x +/- SD), was included as an additional fitted parameter for all of the indicators except water. Using the short catheters, the average DN for the model with delay was 0.31 +/- 0.13 for closed and 0.22 +/- 0.07 for mixed boundary conditions, for all reference indicators. Mean transit times and the variances of the fitted distributions were always smaller than the experimental ones (on average, by 6.8 +/- 3.7% and 58 +/- 19%, respectively). In conclusion, the DM is a reasonable descriptor of dispersion for the early-in-time data and not the late-in-time data. The existence of a common DN for all noneliminated reference indicators suggests that intrahepatic dispersion depends only on the geometry of the vasculature rather than the diffusional processes. The role of the nonsinusoidal ("large") vessels can be partly represented by a simple delay.

Evidence that interference with binding to hepatic cytosol binders can inhibit bile acid excretion in rats

We previously identified that Y' bile acid binders (3alpha- hydroxysteroid dehydrogenases) interact with bile acids in intact rat hepatocytes using [3beta-3H, C24-14C]bile acids and that indomethacin, a competitive inhibitor of 3alpha-hydroxysteroid dehydrogenase, inhibits 3H-loss from the C3-position of bile acids as well as inhibits hepatic bile acid removal and excretion. To study the kinetics of these inhibitory effects, glycocholate transport was studied in the absence and presence of indomethacin in the single-pass perfused rat liver. Indomethacin decreased net hepatic glycocholate uptake in the perfused liver, which was confirmed in isolated hepatocytes and basolateral liver plasma membrane vesicles. However, indomethacin markedly increased the sinusoidal efflux and decreased the biliary excretion of glycocholate in the perfused liver. These observations indicate that the effect of indomethacin to delay biliary glycocholate excretion is related to either intracellular or canalicular glycocholate transport. The latter possibility seemed unlikely because indomethacin did not inhibit electrogenic or adenosine triphosphate (ATP)-dependent glycocholate uptake by canalicular liver plasma membrane vesicles. Thus, the current data support an important role for binding of bile acids to cytosolic proteins in overall hepatic transport and suggest that specific interference with cytosolic binding can interfere with the excretion of bile acids. (Hepatology 1996 Jun;23(6):1642-9)

Evidence That Interference With Binding to Hepatic Cytosol Binders Can Inhibit Bile Acid Excretion in Rats

We previously identified that Y' bile acid binders (3alpha-hydroxysteroid dehydrogenases) interact with bile acids in intact rat hepatocytes using [3beta-3H, C24-14C]bile acids and that indomethacin, a competitive inhibitor of 3alpha-hydroxysteroid dehydrogenase, inhibits 3H-loss from the C3-position of bile acids as well as inhibits hepatic bile acid removal and excretion. To study the kinetics of these inhibitory effects, glycocholate transport was studied in the absence and presence of indomethacin in the single-pass perfused rat liver. Indomethacin decreased net hepatic glycocholate uptake in the perfused liver, which was confirmed in isolated hepatocytes and basolateral liver plasma membrane vesicles. However, indomethacin markedly increased the sinusoidal efflux and decreased the biliary excretion of glycocholate in the perfused liver. These observations indicate that the effect of indomethacin to delay biliary glycocholate excretion is related to either intracellular or canalicular glycocholate transport. The latter possibility seemed unlikely because indomethacin did not inhibit electrogenic or adenosine triphosphate (ATP)-dependent glycocholate uptake by canalicular liver plasma membrane vesicles. Thus, the current data support an important role for binding of bile acids to cytosolic proteins in overall hepatic transport and suggest that specific interference with cytosolic binding can interfere with the excretion of bile acids.

Oleate uptake by isolated hepatocytes and the perfused rat liver is competitively inhibited by palmitate.

Competition for uptake between long-chain free fatty acids has been difficult to document, because there has been no algorithm for computing unbound concentrations of two fatty acids simultaneously in solution with albumin. We modified an iterative procedure to permit this computation and studied initial [3H]oleate uptake by isolated hepatocytes and steady-state uptake by the single-pass perfused rat liver from 600 microM bovine serum albumin solutions containing various concentrations of oleate in the presence and absence of palmitate. In both systems, the Michaelis-Menten constant was significantly higher in the presence of palmitate than in its absence, whereas the maximal reaction velocity was unaltered, indicating competitive inhibition. In additional experiments employing the multiple transhepatic indicator-dilution technique, the influx rate constant and permeability-surface area product for oleate influx were significantly reduced by palmitate, confirming that the competition observed in the conventional perfused liver studies was at the influx step. Long-chain fatty acid uptake has now been shown to exhibit all the kinetic properties of facilitated transport and cannot be attributed solely to passive diffusion.

Characterization of phloretin-sensitive urea export from the perfused rat liver.

In single pass perfused rat liver, rapid osmotic water shifts across the plasma membrane in response to hyperosmolar urea were followed by monitoring liver mass and transient concentrating or diluting effects on Na+ concentration in effluent perfusate. Sudden addition or removal of hyperosmolar urea (200mM, resulting in a step change of the perfusate osmolarity from 305 to 505 mosmol/l) had little effect on liver mass or Na+ activity in the effluent perfusate, suggesting that urea equilibrated at a rate similar to that of water across the liver plasma membrane. When, however, phloretin (0.2mM) was present, sudden addition (removal) of urea (200mM) induced within seconds a marked and transient decrease (increase) of both liver mass and effluent Na+ concentration, suggestive of transient osmotic water shifts out of/into the cells. Although to a lesser extent, comparable effects were induced when urea was added/removed in the presence of the phloretin-related phenol compounds 2,4,6-trihydroxyacetophenone (5mM) and 2,4,5-trihydroxybutyrophenone (5mM). Phloretin-induced inhibition of urea export from livers preloaded with [14C]urea was reversible, and no saturation of urea transport was found at concentrations up to 200mM. In contrast to [14C]urea transport, [3H]water transport across the plasma membrane was not affected by phloretin. The data indicate that urea export across the hepatocyte plasma membrane is almost as fast as water export. The urea transport mechanism is sensitive to phloretin and other phenol compounds, works with high capacity and is distinct from the water-transporting system. The regulation of this putative transport mechanism and its relevance for hepatic nitrogen metabolism remain to be established.

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 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 μmol/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), D 2O (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 (k 1), efflux (k −1) and excretion (k seq) rate constants. Concentration-dependent influx (V max = 83 μmol min −1 g −1 and K m = 3.7 μmol/L), efflux (V max = 15 μmol min 1 g −1 and K m = 1.8 μmol/L), and excretion (V max = 94 μmol min 1 g −1 and K m = 1.8 μmol/L) were obtained for BSPGSH, when K m values are expressed in terms of the unbound concentrations. In these calculations, the observed unbound tissue concentration was not used for estimation of the V max and K m 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 V max of 97 μmol min −1g −1 and a K m of 3.6 μmol/ L, values similar to those obtained from MID. Regression of rates of biliary excretion or net plasma disappearance against the logarithmic average unbound concentration, however, yielded a V max of 50 μmol min -1g −1 and a K m of 3.3 to 3.6 μmol/L, values which were, as expected, different from those obtained with MID for either influx, efflux, or removal. It was found that intracellular BSPGSH levels had not increased sufficiently enough to saturate efflux and excretion, and the limiting feature of the transfer process at high- bulk concentration of BSPGSH appeared to be the influx process.

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 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)

Metabolism of cysteinyl leukotrienes in the perfused rat liver: The influence of endotoxin pretreatment and the cellular hydration state

The influence of endotoxin on the hepatic metabolism and elimination of 3H-leukotriene C4 (LTC4) and 3H-leukotriene E4 was studied in the single-pass perfused rat liver. Endotoxin (4 mg/kg body mass) was injected intraperitoneally 8 to 10 hours before livers were isolated for perfusion. Tritiated leukotriene C4 and leukotriene E4 (10 nmol/L) were infused for 5 minutes, and metabolites in bile were determined by high-pressure liquid chromatography. In livers without endotoxin pretreatment, single-pass uptake of LTC4 was 77.3% +/- 3.2%, and 73.8% +/- 1.8% of the radioactivity taken up was excreted into the bile within 80 minutes. In endotoxin-pretreated livers, LTC4 uptake was 62.8% +/- 3.5% and only 31.2% +/- 1.5% of the radioactivity taken up was eliminated into the bile within 80 minutes. Bile flow was reduced to 0.20 +/- 0.07 microL/min, compared with 1.18 +/- 0.18 microL/g/min in untreated livers. Biliary excretion of infused 3H-LTE4 was also reduced in endotoxin-pretreated livers (31.5% +/- 6.1% compared with 61.4% +/- 3.3% without endotoxin pretreatment), whereas uptake was not significantly different. The effect of cellular hydration state on leukotriene processing was also investigated. Anisoosmotic cell volume changes did not influence uptake and biliary excretion of 3H-LTC4 and its metabolism in control livers. In endotoxin-pretreated livers, however, cell swelling induced by hypotonic perfusion media (225 mOsm/L) or by 3 mmol/L glutamine increased biliary elimination of the radioactivity taken up by 68% and 54%, respectively. Bile flow was also stimulated (0.31 +/- 0.09 microL/g/min and 0.46 +/- 0.01 microL/g/min, respectively).(ABSTRACT TRUNCATED AT 250 WORDS)

Metabolism of cysteinyl leukotrienes in the perfused rat liver: The influence of endotoxin pretreatment and the cellular hydration state

The influence of endotoxin on the hepatic metabolism and elimination of 3H-leukotriene C 4 (LTC 4) and 3H-leukotriene E 4 was studied in the single-pass perfused rat liver. Endotoxin (4 mg/kg body mass) was injected intraperitoneally 8 to 10 hours before livers were isolated for perfusion. Tritiated leukotriene C 4 and leukotriene E 4 (10nmol/L) were infused for 5 minutes, and metabolites in bile were determined by high-pressure liquid chromatography. In livers without endotoxin pretreatment, single-pass uptake of LTC 4 was 77.3% ± 3.2%, and 73.8% ± 1.8% of the radioactivity taken up was excreted into the bile within 80 minutes. In endotoxin-pretreated livers, LTC 4 uptake was 62.8% ± 3.5% and only 31.2% ± 1.5% of the radioactivity taken up was eliminated into the bile within 80 minutes. Bile flow was reduced to 0.20 ± 0.07 μL/g/min, compared with 1.18 ± 0.18 μL/g/min in untreated livers. Biliary excretion of infused 3H-LTE 4 was also reduced in endotoxin-pretreated livers (31.5% ± 6.1% compared with 61.4% ± 3.3% without endotoxin pretreatment), whereas uptake was not significantly different. The effect of cellular hydration state on leukotriene processing was also investigated. Anisoosmotic cell volume changes did not influence uptake and biliary excretion of 3H-LTC 4 and its metabolism in control livers. In endotoxin-pretreated livers, however, cell swelling induced by hypotonic perfusion media (225 mOsm/L) or by 3 mmol/L glutamine increased biliary elimination of the radioactivity taken up by 68% and 54%, respectively. Bile flow was also stimulated (0.31 ± 0.09 μL/g/min and 0.46 ± 0.01 μL/g/min, respectively). Colchicine (5 μmol/L) completely abolished the stimulatory effect of hypotonic perfusion on biliary leukotriene excretion. Hypertonic perfusion decreased leukotriene elimination into bile to 18.1% ± 3.8% of the radioactivity taken up and bile flow to 0.11 ± 0.04 μL/g/min. In livers perfused with hypertonic perfusion buffer, there was a higher amount of polar ω-oxidation metabolites detectable in bile compared with normotonic conditions. The data indicate that endotoxin impairs the canalicular excretion of cysteinyl leukotrienes, whereas there is only minor influence on uptake and intracellular metabolism. Cell swelling increases the biliary elimination of cysteinyl leukotrienes in endotoxin-treated livers, whereas cell shrinkage has the opposite effect, suggesting a modulatory role of cell volume in the canalicular transport of cysteinyl leukotrienes, at least in endotoxemia. Colchicine sensitivity of the cell volume effect suggests the involvement of a microtubule-dependent mechanism.

Sulfation of acetaminophen by the perfused rat liver: The effect of red blood cell carriage

Acetaminophen uptake and conversion in the perfused rat liver to acetaminophen sulfate was studied with the multiple indicator dilution technique (MID). Because acetaminophen is avidly bound to red blood cells and not albumin, a pre-equilibrated MID dose containing the noneliminated references (51Cr-labeled red blood cells [RBC, a vascular reference], [58Co]EDTA [a small molecular weight interstitial reference that does not enter cells], and D2O [a cellular reference]) and [3H]-acetaminophen was introduced into the portal vein of the single-pass perfused rat liver (1 mg/L acetaminophen) under varying conditions of hematocrit, with observation of timed outflow profiles in the hepatic venous blood. The [3H]acetaminophen curve exhibited an early high peak, paralleling that for red cells and varying with hematocrit, followed by a prolonged decline, with the late appearance of acetaminophen sulfate product; the early peak disappeared when red cells were absent from the dose and perfusate. Analysis demonstrated a slow release of acetaminophen from the red blood cells and rapid liver cell entry, so that red cell binding was displayed as a red cell carriage effect that reduced the rate of liver cell entry and hence of sulfation of [3H]-acetaminophen. The liver cells exhibited a concomitant very low permeability to product acetaminophen sulfate, leading to protracted product outflow curves. An inferred slow efflux-mediated storage phenomenon for product was found to evolve as a result.

Sulfation of acetaminophen by the perfused rat liver: The effect of red blood cell carriage

Acetaminophen uptake and conversion in the perfused rat liver to acetaminophen sulfate was studied with the multiple indicator dilution technique (MID). Because acetaminophen is avidly bound to red blood cells and not albumin, a pre-equilibrated MID dose containing the noneliminated references ( 51Cr-labeled red blood cells [RBC, a vascular reference], [ 58co]EDTA [a small molecular weight interstitial reference that does not enter cells], and D 2O [a cellular reference]) and [ 3H]-acetaminophen was introduced into the portal vein of the single-pass perfused rat liver (1 mg/L acetaminophen) under varying conditions of hematocrit, with observation of timed outflow profiles in the hepatic venous blood. The [ 3H]acetaminophen curve exhibited an early high peak, paralleling that for red cells and varying with hematocrit, followed by a prolonged decline, with the late appearance of acetaminophen sulfate product; the early peak disappeared when red cells were absent from the dose and perfusate. Analysis demonstrated a slow release of acetaminophen from the red blood cells and rapid liver cell entry, so that red cell binding was displayed as a red cell carriage effect that reduced the rate of liver cell entry and hence of sulfation of [ 3H]-acetaminophen. The liver cells exhibited a concomitant very low permeability to product acetaminophen sulfate, leading to protracted product outflow curves. An inferred slow efflux-mediated storage phenomenon for product was found to evolve as a result.

Hepatic uptake and biliary excretion of the neuroprotectant 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo-(f)-Ouinoxaline in rat liver: Involvement of an organic anion transport system

The: hepatic uptake and biliary excretion of the neuroprotective drug 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo-(f)-quinoxaline (NBQX) was studied in rat liver. In the isolated single-pass perfused rat liver preparation NBQX was infused in protein-free Krebs-Henseleit bicarbonate buffer at input concentrations ranging from 0.5 to 15 μM. The hepatic uptake could be characterized as a pseudo firstorder unidirectional process with an apparent half-life of 5.8 min. Significantly higher concentrations of NBQX were measured in bile compared to perfusate with a maximal ratio at the lowest input concentration (approx. 2400-fold). Hepatic uptake and biliary excretion of NBQX exhibited saturation at increasing input concentrations, indicating an active transport mechanism. The uptake process could be described by Michaelis-Menten kinetics resulting in a K m,uptake of 2.2 μM. The corresponding maximal uptake rate ( V max,uptake) was calculated to be 103 nmol/min. The maximal biliary excretion rate was estimated to be 58 nmol/min. The rate-limiting factor in the overall hepatic elimination was thus biliary excretion. Co-infusion with the uricosuric drug probenecid resulted in significantly decreased hepatic uptake and biliary excretion. These data suggest that NBQX is eliminated by an organic anion transport system in rat liver which is sensitive to probenecid.