Summary: The isolatedperfusedratliver method (IPRL) was used to find, isolate and identify further metabolites of Phase I and Phase II biotransformation of the potential cytostatic agent benfluron with special regard to the conjugation pro- cesses. Its pharmacokinetic profile during the perfusion was also estimated. The ratliver was isolated from the body and perfused in vitro using a recirculating perfusion system. Benfluron was added to the reservoir as a bolus in doses of 200, 100, 30 mg/kg of body weigh and 1 mg/perfusate volume and also as a continual infusion in a dose of 0.1 mg/min in se- parate series of experiments. The following metabolites formed during Phase I biotransformation were found in the per- fusion liquid as well as in the bile: benfluron N-oxide, 9-hydroxy benfluron, demethylated 9-hydroxy benfluron, demethylated benfluron, and reduced benfluron. The major Phase II metabolite found in the bile samples was the glucu- ronide of 9-hydroxy benfluron. The pharmacokinetic profile of benfluron in IPRL indicated its main disposition and me- tabolic pathway, i.e. its rapid extraction from perfusate by the liver (t 1/2 α = 3.76 min), 9-hydroxylation followed up O-glucuronidation and excretion to the bile. It was revealed that 12 % of the total dose of the parent compound was exc- reted to the bile in the form of conjugates during the first hour of perfusion, 32 % during 1.5 hour, and 70 % during 2 hours after the administration of benfluron. The conjugates with glucuronic acid represented 96-98 % of all metabolites found in the bile.
isolatedperfusedratliver and have compared rates of synthesis in this system with absolute rates of degradation measured in vivo. Rat livers, perfused for 5 h with a recycling fluid consisting of a perfluorochemical emulsion (Fluosol 43), were used to demonstrate a cumulative increase of HF in the perfusate as measured by a specific and sensitive radioimmunoassay. The rate of increase in the perfusate pool of HF during the final 4 h of perfusion yielded a mean synthetic rate of 3.5 micrograms/h per 100 g body wt, which was approximately 0.2% of the synthetic rate of albumin in the same system. The cumulative appearance of albumin and transferrin was linear after 1 h and calculated rates of synthesis were 2,012 micrograms/h per 100 g and 263 micrograms/h per 100 g body wt, respectively. De novo synthesis of HF was confirmed by demonstrating incorporation of [14C]lysine into specific immunoprecipitates of HF, and by the observations that both specific incorporation of labeled amino acid and net release of immunoassayable HF were inhibited by the administration of cycloheximide. Finally, it was evident that the rates of synthesis observed in the isolatedperfusedliver agreed […]
coenzyme A reductase was studied. In liver removed during the basal period of the diurnal cycle of enzyme activity, a 227 +/- 41% increase in enzyme activity occurred after 3 h of a plasma-free perfusion. This could be prevented by the addition of cycloheximide or pure cholesterol (dispersed with lecithin) to the perfusate. In contrast, the continuous addition of taurocholate or taurochenodeoxycholate, alone or in combination, at a variety of rates did not prevent the increase in enzyme activity. The added bile salts were efficiently extracted from the perfusate and excreted in the bile. The addition of these bile salts to a cholesterol- enriched perfusate did not alter the effect obtained with cholesterol alone. If the perfusate contained whole serum, the increase induced by perfusion in the basal period was smaller (88 +/- 27%) than with plasma-free perfusate. Again, the major bile salts of the rat failed to prevent the increase in enzyme activity induced by liver perfusion. If livers were removed and perfused at the height of the diurnal cycle of enzyme activity, the enzyme activity remained high (2 +/- 10% increase) rather than decreasing, as occurs in vivo. If cholesterol was added to these perfusions, a 52 +/- 4% decrease was induced. Bile salt addition induced no […]
Transport of 35S-labeled sulfobromophthalein [35S]BSP was studied in short-term cultured rat hepatocytes incubated in bovine serum albumin. At 37 degrees C, initial uptake of [35S]BSP was 5-10-fold that at 4 degrees C, linear for at least 15 min, saturable, inhibited by bilirubin, and reduced by greater than 70% after ATP depletion or isosmotic substitution of sucrose for NaCl in medium. Replacement of Na+ by K+ or Li+ did not alter uptake,
albumin. Ratliver was perfused with a protein-free fluorocarbon medium, and single-pass uptake of 1, 10, or 200 nmol of [3H]bilirubin was determined after injection as an equimolar complex with 125I-albumin, with 125I-ligandin, or free with only a [14C]sucrose reference. Uptake of 10 nmol of [3H]bilirubin was 67.5 +/- 3.7% of the dose when injected with 125I- albumin, 67.4 +/- 6.5% when injected with 125I-ligandin, and 74.9 +/- 2.4% when injected with [14C]sucrose (P greater than 0.1). At 200 nmol, uptake fell to 46.4 +/- 3.1% (125I-
transplantation? We studied isolatedperfused livers at 10 mM glucose from 27 rats rendered diabetic with streptozotocin and then transplanted with approximately 2 x 10(3) islets, 2 (n = 5), 7 (n = 5), 30 (n = 5), and 200 (n = 12) d after transplantation. 12 out of 12 of the 200-d IHI-Tx secreted insulin in coordinate pulses (frequency 3.9 +/- 0.3 pulses/h,
The hypothesis that intracellular generation of reactive oxygen species in hepatocytes or reticuloendothelial cells may cause ischemia-reperfusion injury was tested in isolatedperfused livers of male Fischer rats. GSSG was measured in perfusate, bile, and tissue as a sensitive index of oxidative stress. After a preperfusion phase of 30 min, the perfusion was stopped (global ischemia) for various times (30, 120 min) and the liver was reperfused for another 60 min. The bile flow (1.48 +/- 0.17 microliters/min X gram liver weight), the biliary efflux of total glutathione (6.54 +/- 0.94 nmol GSH eq/min X g), and GSSG (1.59 +/- 0.23 nmol GSH eq/min X g) recovered to 69-86% after short-term ischemia and to 36-72% after 2 h of ischemia when compared with values obtained from control livers perfused for the same period of time. During reperfusion, the sinusoidal efflux of total glutathione (16.4 +/- 2.1 nmol GSH eq/min X g) and GSSG (0.13 +/- 0.05 nmol GSH eq/min X g) did not change except for an initial 10-30-s increase during reperfusion washout. No increased GSSG secretion into bile was detectable at any time during reperfusion. The liver content of total glutathione (32.5 +/- 3.5 nmol GSH eq/mg protein) and GSSG (0.27 +/- 0.09 nmol GSH eq/mg protein) did not change significantly during any period of ischemia or reperfusion. We […]
contribution of transcellular vesicular transport (transcytosis) to the blood-to-bile movement of inert fluid-phase markers of widely varying molecular weight. First, under steady-state conditions, the perfusedratliver secreted even large markers in appreciable amounts. The bile-to-plasma (B/P) ratio of these different markers, including microperoxidase (B/P ratio = 0.06; mol wt = 1,879), insulin (B/P ratio = 0.09, mol wt = 5,000), horseradish peroxidase (B/P ratio = 0.04, mol wt = 40,000), and dextran (B/P ratio = 0.09, mol wt = 70,000), exhibited no clear ordering based on size alone, and when dextrans of two different sizes (40,000 and 70,000 mol wt) were studied simultaneously, the relative amounts of the two dextran
Since ranolazine inhibits oxygen uptake, it seems reasonable to assume that this also represents diminished mitochondrial ATP production. Corroborating this, a diminution of the ATP content was found in the perfusedliver of fasted rats and under gluconeogenic conditions. However, no such diminution was found in the liver of fed rats under glycogenolytic and glycolytic conditions. The fact that the ATP content was not affected by rano- lazine in the fed state is most probably an event caused by the compensatory ATP production in the glycolytic pathway, which is stimulated by ranolazine (Figure 1). Actually, in the case of the experiments shown in Figure 1, in which 100 µM ranolazine was infused, the excess lactate production was approximately equal to 0.7 µmol min −1 ∙g −1 . On stoichiometric grounds, this corresponds also to a net ATP production rate of 0.7 µ mol∙min −1 ∙g −1 . The diminution of oxygen uptake, on the other hand, was around 0.1 µmol O 2 min −1 ∙g −1 (or 0.2 µg-atom oxygen
disappearance of 125I-TGF beta by 80%. 60 min after intrafemoral injection, 63% of the recovered label was present in liver and/or bile; by 90 min, most of the label removed by the liver (83%) had been slowly excreted into bile. Nearly all the label in bile (96%) was soluble in trichloracetic acid and not immunoprecipitable by specific antiserum. Colchicine and vinblastine inhibited cumulative biliary excretion of label by 28 and 37%, respectively; chloroquine and leupeptin each increased the amount of label in bile that was precipitable by trichloracetic acid and that coeluted with authentic 125I-TGF beta on molecular sieve chromatography. There was efficient first-pass hepatic extraction of 125I-TGF beta (36%) in the isolatedperfusedratliver, which was inhibited by unlabeled TGF beta (but not by epidermal growth factor, EGF) and by lectins in a dose-dependent manner; prolonged fasting also decreased clearance (26%). After fractionation of liver by differential or isopycnic centrifugation, radiolabel codistributed with […]
To assess the effects of tauroursodeoxycholic acid (TUDCA) on bile excretory function, we examined whether TUDCA modulates vesicular exocytosis in the isolatedperfusedliver of normal rats in the presence of high (1.9 mM) or low (0.19 mM) extracellular Ca++ and in cholestatic rats 24 h after bile duct ligation. In addition, the effects of TUDCA on Ca++ homeostasis were compared in normal and in cholestatic hepatocytes. In the isolatedperfusedratliver, TUDCA (25 microM) stimulated a sustained increase in the biliary
We studied the effect of DL-3-hydroxybutyrate and acetoacetate on lactate transport into isolated hepatocytes and on lactate removal in the isolatedperfusedratliver. Ketone bodies inhibited lactate transport into isolated hepatocytes (maximum, 35% at concentrations of 10- 20 mM). Lactate removal and glucose production by perfused livers were examined before and after the introduction of a constant infusion of hydroxybutyrate, acetoacetate, or
The pathogenesis of liver disease in protoporphyria has been presumed to result from the hepatic deposition of protoporphyrin. To examine the effects of protoporphyrin on hepatic bile flow and histopathology, studies were performed employing an isolated, in situ, ratliver perfusion system. Rat livers in the control group were perfused with 0-80 µmol sodium taurocholate/h. Rat livers in the experimental group were perfused with sodium taurocholate and (a) sufficient quantities of protoporphyrin to produce maximal canalicular secretion and (b) perfusate protoporphyrin concentrations of 0.01, 0.1, and 1 µM.
dibromosulphthalein (DBSP). In this paper, the hepatobiliary transport of glutathione and a glutathione conjugate was studied in normal Wistar rats and TR- rats. It was shown that glutathione is virtually absent from the bile of TR- rats. In the isolated, perfusedliver the secretion of glutathione and the glutathione conjugate, dinitrophenyl-glutathione (GS-DNP), from hepatocyte to bile is severely impaired, whereas the sinusoidal secretion from liver to blood is not affected. The secretion of GS-DNP was also studied in isolated hepatocytes. The secretion of GS-DNP from cells isolated from TR- ratliver was significantly slower than from normal hepatocytes. Efflux of GS-DNP was a saturable process with respect to
Rat kidneys perfused outside of the body with an artificial medium are able to increase their fractional excretion of potassium in response to a rising concentration of potassium in the medium but never show net secretion of potassium. By contrast, isolatedperfused kidneys from chronically potassium-loaded rats regularly secrete potassium in excess of the amount filtered. Ouabain completely blocks the secretion of potassium by these isolated kidneys, suggesting that Na-K-ATPase mediates potassium secretion by potassium-adapted rats. Neither sodium deprivation, pretreatment with deoxycorticosterone, nor pretreatment with methylprednisolone prepared the kidney to secrete potassium, despite stimulation of Na-K- ATPase activity in cortex or outer medulla. Potassium loading was the only maneuver tested that increased the activity of Na-Katpase in the inner medulla (white papilla) and also produced potassium secretion by the isolated kidney. Surgical ablation of the papilla