Hepatic uptake of polystyrene microspheres in rats: effect of particle size on intrahepatic distribution.

The in vivo disposition of polystyrene microsphere (MS) with the particle size of 50 nm (MS-50) or 500 nm (MS-500) was characterized after intravenous administration to rats. A rapid elimination from systemic circulation was observed for both MSs. Tissue distribution of MS-50 and MS-500 at 1 h after intravenous injection indicated that both MSs were exclusively distributed to liver and that small but significant amounts of MS-50 and MS-500 were also distributed to lung and spleen, respectively. To investigate the intrahepatic distribution of MS, liver was separated into liver parenchymal cells (PC) and non-parenchymal cells (NPC) at 1 or 6 h after intravenous administration. The contribution of each cell fraction was dependent on both the size of MS and the time after administration. Furthermore, by separating the NPC into endothelial cells and Kupffer cells using a centrifugal elutriation method, their contribution was also evaluated. For both MSs, Kupffer cells were recognized to be mostly responsible for the hepatic uptake, although a significant amount of MS-50 (about 28% of total uptake) was taken up by PC. On the other hand, there was little contribution of PC (about 5%) to the hepatic uptake of MS-500. The endothelial cells were contributed larger to the uptake of MS-500 (about 24%) than that of MS-50 (13%).     

Pre-coating with serum albumin reduces receptor-mediated hepatic disposition of polystyrene nanosphere: implications for rational design of nanoparticles

We evaluated the in vivo disposition characteristics of polystyrene nanospheres (NS) with the particle size of 50 nm (NS-50) pre-coated with human serum albumin (HSA) after intravenous administration in rats. HSA-coated NS-50 showed much longer blood-circulating property and the hepatic uptake clearance for HSA-coated NS-50 was about 1/5 of that for NS-50. In parallel with the results obtained in the in vivo study, liver perfusion experiments also showed that the hepatic disposition of HSA-coated NS-50 was much less than that of NS-50 in the presence of serum in the perfusate. To unravel the mechanism behind the less affinity of HSA-coated NS-50 to the liver, serum proteins associated on the surface was quantitatively and qualitatively assessed. The results indicated that pre-coated HSA impaired subsequent association of serum proteins onto the surface, suggesting that the association of a given serum protein with opsonic activity might be suppressed by HSA pre-coating. From these findings, pre-coating of nanoparticles with serum albumin could be useful to prevent their rapid clearance by mononuclear phagocyte system in vivo.     

Surface hydrophobicity of particles is not necessarily the most important determinant in their in vivo disposition after intravenous administration in rats.

The in vivo disposition of polystyrene microsphere (MS) with the particle size of 50 nm (MS-50) and lecithin-coated MS-50 (LMS-50) after intravenous administration to rats was characterized. While a rapid elimination from the systemic circulation was observed for MS-50, much more prolonged circulating property was observed for LMS-50. In addition, this in vivo disposition property of LMS-50 was suggested to be ascribed to its lower affinity to the liver, which is the determining organ of the in vivo disposition of MS-50. The evaluation of surface hydrophobicity of MS-50 and LMS-50 in buffer solution revealed that the surface of MS-50 is more hydrophobic than that of LMS-50. However, LMS-50 was oppositely found to be more hydrophobic than that of MS-50 in rat serum. The profiles of serum proteins associated with MS-50 and LMS-50 were also examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The results showed that the amounts of some adsorbed proteins are greatly different between MS-50 and LMS-50. From these findings, it was suggested that the substantial difference in the in vivo disposition between MS-50 and LMS-50 would not be attributed to the difference in their surface hydrophobicity in the blood, but the difference in the type of serum proteins associated with them.     

Important role of serum proteins associated on the surface of particles in their hepatic disposition.

To elucidate the important factors for the difference in the hepatic disposition between polystyrene nanospheres with a size of 50 nm (NS-50) and lecithin-coated NS-50 (LNS-50), the liver perfusion studies and the in vitro uptake studies using the cultured Kupffer cells were performed. It was suggested that opsonin-mediated phagocytosis is not significantly involved in the hepatic disposition of LNS-50 in the presence of serum, whereas its involvement in the hepatic uptake of NS-50 was clearly demonstrated. Western blot analysis showed that IgG, complement C3, and fibronectin, well-known opsonins in the serum, adsorbed on the surface of NS-50 in larger amount than on the surface of LNS-50. On the other hand, serum albumin, which was suggested to function as a dysopsonin for the hepatic disposition of NS-50, was associated with both spheres almost to the same extent. These findings suggest that the hepatic disposition of LNS-50 at lower level should be ascribed to the less amount of serum opsonins associated on the surface and that the serum proteins associated with these spheres should be important as a determinant for their hepatic disposition.     

Comparison of vanadate and ouabain effects on liver hemodynamics and bile production in the perfused rat liver

Cumulative addition of vanadate or ouabain to rat liver perfusions giving perfusate concentrations up to about 500 microM revealed that both liver hemodynamics and bile production are influenced by these substances, but their ways of action differed markedly. Vanadate increased hepatic vascular resistance in a dose-dependent manner with an apparent Km of 30 microM. Not until vanadate concentrations in perfusate reached 60 to 70 microM did the hepatic oxygen consumption decrease significantly together with a decrease in bile flow. Ouabain at perfusate concentrations up to nearly 300 microM caused only a slight fall in perfusate flow, a dose-dependent slight fall in oxygen uptake and a dose-dependent, marked increase in bile flow. Further addition of even small amounts of ouabain initiated a marked fall in perfusate flow, oxygen uptake and bile production and appeared to induce maldistribution of intrahepatic perfusate flow. The vasoconstrictive effect of vanadate was not influenced by alpha or beta blockers, atropine or blockers of Ca++-transport, whereas the effect of ouabain could be strongly reduced by phenoxybenzamine or verapamil. Vanadate-induced vasoconstriction may be caused by an inhibition of smooth muscle Ca++-adenosine triphosphatase and ouabain may induce vasoconstriction by inhibiton of smooth muscle Na, K-adenosine triphosphatase. The hepatic uptake and excretion of ouabain may explain the choleresis observed at small perfusate concentrations of ouabain. Inhibition of bile production at higher concentrations of ouabain and vanadate could be secondary to simultaneous changes in liver hemodynamics.     

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.     

Evaluation of route of input on the hepatic disposition of diazepam

Diazepam, a drug of high intrinsic clearance, was studied in the in situ rat liver dually perfused with Krebs-bicarbonate buffer containing human serum albumin (HSA; 0-1%) and unlabeled diazepam (1 mg/l) under constant hepatic arterial (3 ml/min) and portal venous (PV; 12 ml/min) flow rates. Events after a unit impulse (using [(14)C]diazepam) and at steady state (using unlabeled diazepam) were evaluated. In the absence of HSA the fractional effluent recovery (F) after hepatic arterial infusion (0.046 +/- 0.013) was about twice that after PV infusion (0.019 +/- 0.006). With HSA present, regardless of input route, F increased as unbound diazepam fraction in perfusate decreased (e.g., for PV, F = 0.58 +/- 0.05 and 0.69 +/- 0.02 for unbound diazepam fraction values of 0.18 +/- 0.01 and 0.037 +/- 0.01 at 0.25% and 1% HSA). The effluent [(14)C]diazepam profile was also dependent upon HSA. On decreasing HSA from 1 to 0.25% the early sharp peak (at 12-20 s) was replaced by a flatter unimodal profile with a later peak (at 60-80 s). Comparison of estimated effective permeability-surface area product to perfusate flow ratios (4.4 for 1% HSA and 21 for 0.25% HSA) indicated a shift from a perfusion rate-limited uptake with 0.25% HSA to one intermediate between permeability and perfusion at 1% HSA. Recognizing that orally absorbed drug enters the liver only via PV and i.v. drug via both vascular routes, this study emphasizes the difference in hepatic extraction of compounds depending on route of input, and the role that alteration in perfusate binding has on hepatic drug disposition.     

Ex situ inhibition of hepatic uptake and efflux significantly changes metabolism: hepatic enzyme-transporter interplay

The disposition of digoxin and the influence of the organic anion transporting polypeptide (Oatp)2 inhibitor rifampicin and the P-glycoprotein (P-gp) inhibitor quinidine on its hepatic disposition were examined in the isolated perfused rat liver. Livers from groups of rats were perfused in a recirculatory manner after a bolus dose of digoxin (10 microg), a dual substrate for Oatp2 and P-gp as well as CYP3A. Perfusions of digoxin were also examined in groups of rats in the presence of the inhibitors: rifampicin (100 microM) or quinidine (10 microM). In all experiments, perfusate samples were collected for 60 min. Digoxin and its primary metabolite were determined in perfusate and liver by liquid chromatography/mass spectrometry. The area under the curve (AUC) from 0 to 60 min was determined. The AUC +/- S.D. of digoxin was increased from control (3880 +/- 210 nM x min) by rifampicin (5200 +/- 240 nM x min; p < 0.01) and decreased by quinidine (3220 +/- 340 nM x min; P < 0.05). It is concluded that rifampicin limits the hepatic entrance of digoxin and reduced the hepatic exposure of digoxin to CYP3A by inhibiting the basolateral Oatp2 uptake transport, whereas quinidine increased the hepatic exposure of digoxin to CYP3A by inhibiting the canalicular P-gp transport. These data emphasize the importance of uptake and efflux transporters on hepatic drug metabolism.     

Pharmacokinetic analysis of in vivo disposition of succinylated proteins targeted to liver nonparenchymal cells via scavenger receptors: importance of molecular size and negative charge density for in vivo recognition by receptors

In vivo disposition characteristics of succinylated (Suc-) proteins were studied after intravenous injection in mice in relation to their molecular characteristics as negatively charged macromolecules. Recombinant superoxide dismutase (SOD; molecular mass, 32 kDa), bovine serum albumin (BSA; molecular mass, 67 kDa), and bovine IgG (molecular mass, 150 kDa) were used to produce succinylated derivatives with different degrees of modification. (111)In-labeled Suc-SODs were rapidly excreted into the urine with no significant hepatic uptake. In contrast, (111)In-Suc-BSA and Suc-IgG were significantly taken up by liver nonparenchymal cells via scavenger receptors (SRs) according to the degree of succinylation and the dose injected. Interestingly, highly succinylated BSAs exhibited significant accumulation in the kidney at higher doses when the hepatic uptake was saturated. Pharmacokinetic analysis demonstrated that the hepatic uptake of succinylated proteins depended on the molecular size and the estimated surface density of succinylated amino residues. Further analysis based on a physiological pharmacokinetic model, involving a saturable process with Michaelis-Menten kinetics, revealed that the surface density of negative charges was correlated with the affinity of larger succinylated proteins for the hepatic SRs. Thus, the present study has provided useful basic information for a therapeutic strategy and the molecular design of succinylated proteins for use as drug carriers and therapeutic agents per se for SR-mediated targeting in vivo.     

Clearance of Immunoreactive Somatostatin by Perfused Rat Liver

Other investigators have demonstrated that concentrations of immunoreactive somatostatin (IRS) are higher in blood from the hepatic portal vein or its tributaries than in blood from the hepatic or peripheral systemic veins of man and animals. This suggests that there is hepatic extraction of IRS from the portal system in vivo. In the rat, portal vein plasma IRS is reported to be heterogeneous and to contain, in part, a 1,600 mol wt form of IRS which is immunochemically similar to synthetic somatostatin and not significantly bound to high molecular weight plasma protein. Our study was undertaken to determine directly whether unbound synthetic cyclic somatostatin was cleared by the rat liver perfused through the hepatic portal vein in vitro with a recirculating, plasma-free, erythrocyte-containing perfusate.At 37 degrees C and pH 7.40, perfusate IRS, at initial concentrations (1,728 pg/ml) within the range previously reported in rat portal venous blood, was removed by the liver at a rate commensurate with first-order kinetics. Hepatic clearance was 0.84+/-0.04 ml/min per g postperfusion wet weight (SE). Hepatic extraction was 36+/-2%, and t((1/2)) was 20.0+/-1.3 min. Recovery of IRS from the perfusate without the liver was >85%, excluding significant degradation by the medium. Clearance, extraction, and t((1/2)) of IRS were not changed by an unphysiologic IRS concentration (621,500 pg/ml), or by pharmacologic concentrations of insulin (8.2 muM) or glucagon (2.9 muM).The t((1/2)) was prolonged significantly to 28.2+/-1.9 and 45.6+/-4.7 min during perfusions at liver temperatures of 25 degrees and 16 degrees C, respectively. At 37 degrees C, the t((1/2)) was also significantly increased to 28.7+/-3.2 and 24.2+/-1.1 min at perfusate pH 7.06 and 6.78, respectively.These studies indicate that the rat liver clears unbound IRS from the perfusate by a first-order kinetic process that is (a) unsaturable at pharmacologic concentrations, (b) temperature-sensitive and, to a lesser extent, influenced by lowered pH, and (c) not affected by insulin and glucagon. The liver would appear to play an important role in the metabolism of the 1,600 mol wt form of somatostatin. Clearance of endogenous IRS by the liver should be considered in the interpretation of IRS concentrations in the peripheral systemic veins.     

Vitamin D3 uptake by the isolated perfused rat liver from lipoprotein fractions is separate from cholesterol and triglyceride uptake

Using the isolated perfused rat liver we examined the uptake of [14C] or [3H] vitamin D3 and [14C] triglycerides or [3H] cholesterol by the liver of normal rats, from different lipoprotein fractions, as measured by disappearance from the perfusate. When incorporated into chylomicrons only 43% of the vitamin D3 remained in the perfusate at 60 min (i.e. 57% uptake) as compared to 68% of the triglycerides (i.e. 32% uptake). When added on very low density lipoproteins (VLDL) at 60 min 37 +/- 3% (n = 6) of the vitamin D3, 38 +/- 2% of the cholesterol (n = 3) (P NS), and 83 +/- 4% of the triglycerides (n = 3) remained in the perfusate (P less than 0.0005 for cholesterol:triglycerides and vitamin D3:triglycerides). For high density lipoprotein fraction (HDL) isolated perfused livers were studied with and without albumin present in the perfusate. Without albumin 19 +/- 8% (n = 3) of the vitamin D3 and 43 +/- 8% (n = 3) of the cholesterol remained in the perfusate at 60 min. The results with albumin present were 40 +/- 1% (n = 5) of the vitamin D3 and 63 +/- 4% (n = 5) of the cholesterol at 60 min (P less than 0.0005). The cholesterol:cholesterol ester ratio of the VLDL fraction was 8.8:1 and of the HDL fraction 1:1.4. There was no metabolism of vitamin D3 during the 1 h perfusion. These results suggest that vitamin D3 is in equilibrium with the lipoprotein surface, and the hepatic uptake of vitamin D is a surface phenomenon independent of lipoprotein metabolism.     

In vitro uptake of polystyrene microspheres: effect of particle size, cell line and cell density.

Uptake of polycation-DNA particles is the first step in achieving gene delivery with non-viral vehicles. One of the important characteristics determining uptake of DNA particles is their size. Here we have characterized the ability of several cell lines to internalise labelled polystyrene microspheres of different sizes. All the cell lines tested ingested 20-nm microspheres avidly. With larger microspheres (93, 220, 560 and 1010 nm) cell type as well as growth related differences were observed. Whereas some cell lines (HUVEC, ECV 304 and HNX 14C) took up microspheres up to 1010 nm even when the cells were confluent, others did not take up many microspheres larger than 93 nm (Hepa 1-6 and HepG2). In one cell line (KLN 205), uptake of 93-, 220- and 560-nm microspheres was avid in growing cells, but not detectable when they were confluent. In KLN 205 cells, a good correlation was found between the uptake of 560-nm microspheres and the uptake of a peptide-DNA polyplex formulation, when it was prepared under conditions leading to small particle sizes. Little correlation was found when the polyplex formulation was allowed to aggregate.     

O2-dependent hepatotoxicity due to ethylhexanol in the perfused rat liver: mitochondria as a site of action

Toxicity of 2-ethylhexanol, a metabolite of diethylhexyl phthalate, was assessed in the perfused rat liver. Livers from starved rats were perfused with ethylhexanol (3 mM) dissolved in Krebs-Henseleit buffer (pH 7.4, 37 degrees C) saturated with 95% O2-5% CO2 in both the anterograde and retrograde direction. Following infusion of ethylhexanol, O2 uptake and ketone body formation were diminished by 50 and 80%, respectively, and cell damage, as assessed by the appearance of lactate dehydrogenase in the effluent perfusate, was apparent. Both inhibition of O2 uptake by ethylhexanol and the appearance of lactate dehydrogenase in the perfusate were dose-dependent. Only O2-rich upstream regions of the liver lobule were damaged as reflected by trypan blue uptake. Inhibition of O2 uptake by ethylhexanol was also reflected by a 60% decrease in the ATP/ADP ratio. Local rates of O2 uptake, measured using miniature electrodes placed on the liver surface, indicated that ethylhexanol only diminished O2 uptake in O2-rich upstream regions of the liver lobule regardless of the direction of flow. This phenomenon apparently can be explained by a direct effect of ethylhexanol on mitochondria in upstream regions since active state 3 rates of respiration were inhibited by ethylhexanol in isolated mitochondria. Ethylhexanol also caused a dose-dependent decrease in the mitochondrial membrane potential and an increase in the beta-hydroxybutyrate/acetoacetate (B/A) ratio. However, infusion of radical scavengers such as allopurinol, cianidanol and uric acid did not alter lactate dehydrogenase release due to ethylhexanol. Thus, the toxicity of ethylhexanol in the liver is dependent on local O2 tension and mitochondrial are primary targets.(ABSTRACT TRUNCATED AT 250 WORDS)     

Multiple transporters affect the disposition of atorvastatin and its two active hydroxy metabolites: application of in vitro and ex situ systems

Atorvastatin (ATV) is primarily metabolized by CYP3A in the liver to form two active hydroxy metabolites. Therefore, the sequential transport system governed by hepatic uptake and efflux transporters is important for the drug disposition and metabolism. Here, we assessed the interaction of ATV with hepatic uptake transporter organic anion transporting polypeptide (Oatp) and efflux transporter multidrug resistance associated protein 2 (MRP2/Mrp2) in vitro and ex situ using the isolated perfused rat liver (IPRL). Rifampicin (RIF) was chosen as an inhibitor for Oatp in both uptake and IPRL studies. Its inhibitory effects on MRP2 and metabolism were also tested using MRP2-overexpressing cells and rat microsomes, respectively. Our results indicate that RIF effectively inhibits the Oatp-mediated uptake of ATV and its metabolites. Inhibition on MRP2-mediated efflux of ATV was also observed at a high RIF concentration. Compared with ATV alone in the IPRL, the area under the curve(s) (AUC) of ATV was significantly increased by RIF, whereas the AUC of both metabolites were also increased in a concentration-dependent manner. However, the extent of metabolism was significantly reduced, as reflected by the reduced amounts of metabolites detected in RIF-treated livers. In conclusion, inhibition of Oatp-mediated uptake seems to be the major determinant for interaction between ATV and RIF. Metabolites of ATV were subject to Oatp-mediated uptake as well, suggesting that they undergo a similar disposition pathway as the parent drug. These data emphasize the relevance of uptake transporter as being one of the major players in hepatic drug elimination, even for substrates that undergo metabolism.     

Hepatic disposition of the acyl glucuronide 1-O-gemfibrozil-beta-D-glucuronide: effects of clofibric acid, acetaminophen, and acetaminophen glucuronide

Glucuronidation of carboxylic acid compounds results in the formation of electrophilic acyl glucuronides. Because of their polarity, carrier-mediated hepatic transport systems play an important role in determining both intra- and extrahepatic exposure to these reactive conjugates. We have previously shown that the hepatic membrane transport of 1-O-gemfibrozil-beta-D-glucuronide (GG) is carrier-mediated and inhibited by the organic anion dibromosulfophthalein. In this study, we examined the influence of 200 microM acetaminophen, acetaminophen glucuronide, and clofibric acid on the disposition of GG (3 microM) in the recirculating isolated perfused rat liver preparation. GG was taken up by the liver, excreted into bile, and hydrolyzed within the liver to gemfibrozil, which appeared in perfusate but not in bile. Mean +/- S. D. hepatic clearance, apparent intrinsic clearance, hepatic extraction ratio, and biliary excretion half-life of GG were 10.4 +/- 1.4 ml/min, 94.1 +/- 17.9 ml/min, 0.346 +/- 0.046, and 30.9 +/- 4.9 min, respectively, and approximately 73% of GG was excreted into bile. At the termination of the experiment (t = 90 min), the ratio of GG concentrations in perfusate, liver, and bile was 1:35:3136. Acetaminophen and acetaminophen glucuronide had no effect on the hepatic disposition of GG, suggesting relatively low affinities of acetaminophen conjugates for hepatic transport systems or the involvement of multiple transport systems for glucuronide conjugates. In contrast, clofibric acid increased the hepatic clearance, extraction ratio, and apparent intrinsic clearance of GG (P <.05) while decreasing its biliary excretion half-life (P <.05), suggesting an interaction between GG and hepatically generated clofibric acid glucuronide at the level of hepatic transport. However, the transporter protein(s) involved remains to be identified.     

Hepatic disposition and effects of nitric oxide donors: rapid and concentration-dependent reduction in the cytochrome P450-mediated drug metabolism in isolated perfused rat livers

Various mechanisms, including high levels of cytokines and nitric oxide (NO), have been proposed as mediators for inflammation-induced cytochrome 450 down-regulation. However, the contribution of each of these mediators to the observed effects is controversial. We used an isolated perfused rat liver (IPRL) model to test the direct effects of NO donors on CYP450 down-regulation in the absence of cytokines or other confounding in vivo factors. Our hypothesis was that NO rapidly and concentration-dependently decreases CYP450 activities in IPRL. Livers were perfused (60 min) with 50 to 500 microM sodium nitroprusside (SNP) or 100 to 500 microM isosorbide dinitrate (ISDN) as NO donors, and the perfusate and biliary disposition of SNP, ISDN, and generated nitrate/nitrite (NO(x)) were determined. Additionally, at the end of perfusion, catalytic activities and protein levels of various cytochrome isoenzymes were measured. Both SNP and ISDN exhibited linear hepatic disposition with extraction ratios of approximately 0.30 and 0.50, respectively. Furthermore, although in small amounts, both NO donors and NO(x) were found in the bile. Except for CYP2D1, the catalytic activities of all the studied isoenzymes were substantially (up to 85%) decreased by both NO donors. However, the apoprotein levels of isoenzymes remained largely unchanged. Additionally, the inhibitory effects of NO donors were concentration-dependent, with the concentrations of SNP producing one-half of maximum inhibition being in the order of 2C11 > 2B1/2 > 2E1 = 3A2 > 1A1/2. These studies indicate that the effects of NO on the down-regulation of cytochrome 450 catalytic activity are rapid, concentration-dependent, and isoenzyme-selective.     

Effects of ion substitution on bile acid-dependent and -independent bile formation by rat liver.

To characterize the transport mechanisms responsible for formation of canalicular bile, we have examined the effects of ion substitution on bile acid-dependent and bile acid-independent bile formation by the isolated perfused rat liver. Complete replacement of perfusate sodium with choline and lithium abolished taurocholate-induced choleresis and reduced biliary taurocholate output by greater than 70%. Partial replacement of perfusate sodium (25 of 128 mM) by choline reduced bile acid-independent bile formation by 30% and replacement of the remaining sodium (103 mM) by choline reduced bile acid-independent bile formation by an additional 64%. In contrast, replacement of the remaining sodium (103 mM) by lithium reduced bile acid-independent bile formation by only an additional 20%, while complete replacement of sodium (128 mM) by lithium reduced bile formation by only 17%, and lithium replaced sodium as the predominant biliary cation. Replacement of perfusate bicarbonate by Tricine, a zwitterionic amino acid buffer, decreased bile acid-independent bile formation by greater than or equal to 50% and decreased biliary bicarbonate output by approximately 60%, regardless of the accompanying cation. In separate experiments, replacement of sodium by lithium essentially abolished Na,K-ATPase activity measured either as ouabain-suppressible ATP hydrolysis in rat liver or kidney homogenates, or as ouabain-suppressible 86Rb uptake by cultured rat hepatocytes. These studies indicate that bile acid(taurocholate)-dependent bile formation by rat liver exhibits a specific requirement for sodium, a finding probably attributable to the role(s) of sodium in hepatic sodium-coupled taurocholate uptake and/or in maintenance of Na,K-ATPase activity. The surprising finding that bile acid-independent bile formation was substantially unaltered by complete replacement of sodium with the permeant cation lithium does not appear to be explained by Na,K-ATPase-mediated lithium transport. Although alternative interpretations exist, this observation is consistent with the hypothesis that much of basal bile acid-independent bile formation is attributable to an ion pump other than Na,K-ATPase, which directly or indirectly mediates bicarbonate transport.     

Regulation of the Conversion of Thyroxine to Triiodothyronine in the Perfused Rat Liver

This study was undertaken to determine what factors control the conversion of thyroxine (T(4)) to triiodothyronine (T(3)) in rat liver under conditions approximating those found in vivo. Conversion of T(4) to T(3) was studied in the isolated perfused rat liver, a preparation in which the cellular and structural integrity is maintained and that can perform most of the physiologic functions of the liver. The perfused liver readily extracted T(4) from perfusion medium and converted it to T(3). Production of T(3) by the perfused liver was a function of the size of the liver, the uptake of T(4) by the liver, and the presence of T(4)-5'-deiodinase activity. Production of T(3) was increased by increasing the uptake of T(4) by liver, which could be accomplished by increasing the liver size, by increasing the perfusate T(4) concentration, or by decreasing the perfusate albumin concentration. These changes occurred without altering the conversion of T(4) to T(3). The liver had a large capacity for extracting T(4) and for T(4)-5'-deiodination to T(3), which was not saturated at a T(4) concentration of 60 mug/dl. Production of T(3) was decreased by inhibiting hepatic T(4)-5'-deiodinase with propylthiouracil, which decreased T(3) production by decreasing the conversion of T(4) to T(3). Propylthiouracil did not alter hepatic T(4) uptake.Fasting resulted in a progressive decrease in hepatic T(4) uptake to 42% of control levels by the 3rd d of fasting; this was accompanied by a proportionate decrease in T(3) production. The rate of conversion of T(4) to T(3) did not change during fasting. When T(4) uptake in 2-d-fasted rat livers was raised to levels found in fed rats by increasing the perfusate T(4) concentration from 10 to 30 mug/dl, T(3) production returned to normal. Again, no change in the rate of conversion of T(4) to T(3) was observed.These results indicate that the decreased hepatic T(3) production during fasting primarily results from decreased hepatic uptake of T(4), rather than from changes in T(4)-5'-deiodinase activity. Thus, these studies have delineated a new mechanism that functions independently of enzyme quantity or activity whereby production of T(3) from T(4) is regulated.     

Reduced liver uptake of arterially infused melphalan during retrograde rat liver perfusion with unaffected liver tumor uptake

Isolated hepatic perfusion (IHP) with melphalan is used for patients with nonresectable metastases confined to the liver. To improve the efficacy of IHP and to reduce the toxicity to the liver, reversion (retrograde perfusion) of the bloodstream through the liver in a rat model was studied. For liver tumor induction male WAG/Rij rats were inoculated with CC531 cells, a colorectal tumor cell line. After 11 to 12 days the tumor-bearing rat livers were perfused by single-pass perfusion through either the portal (orthograde) or caval vein (retrograde) for different time periods. During perfusion melphalan (160 Schultze) was infused in the hepatic artery. Melphalan concentrations were measured by high-performance liquid chromatography. A rapid extraction of melphalan by the liver occurred in the first 5 min, reaching steady state after 10 to 20 min for both perfusion directions. The melphalan concentration of the outflow perfusate was significantly higher in the retrograde perfusion compared with the orthograde perfusion. The melphalan content of the tumor tissue was unaffected by perfusion direction at any time point. To the contrary, the melphalan uptake in liver tissue was strongly influenced: the melphalan content after 40-min retrograde perfusion was 12% of that after orthograde perfusion. The average tumor/liver concentration ratio was 6 for orthograde perfusion and 30 for retrograde perfusion. In conclusion, retrograde IHP with continuous melphalan infusion in the hepatic artery provides a high tumor uptake of melphalan with potentially reduced liver toxicity compared with orthograde IHP.     

Specific binding of acidic isoferritins to erythroleukemia K562 cells

An investigation of ferritin binding by human erythroleukemia K562 cells was prompted by recent studies suggesting that acidic isoferritins may act as regulators of granulopoiesis and hemopoiesis. Purified human heart and liver ferritins were labeled with iodine 125 and incubated with K562 cells at 37 degrees C. Specific uptake was calculated from the reduction in labeled ferritin binding in the presence of a 1000-fold excess of unlabeled ferritin. Specific uptake of 125I-labeled heart ferritin increased progressively, reaching a maximum after 2 to 3 hours' incubation, although nonspecific binding was too high to derive an affinity constant. There was no specific binding with 125I-labeled liver ferritin, and K562 cells bound neither 125I-labeled human serum albumin nor free 125I. Uptake of heart ferritin was negligible at 4 degrees C and was sharply reduced in the presence of 10% human plasma or fetal calf serum. There was no apparent relationship between the number of days of subculture and the level of uptake of acidic isoferritins by whole cells. These studies demonstrate a selective binding mechanism for acidic isoferritins on erythroleukemia cells and imply that these isoferritins have additional functions besides the storage of iron.