Isolated Rat Liver Preparation Research Articles

Pharmacokinetic analysis of factors determining elimination pathways for sulfate and glucuronide metabolites of xenobiotics II: Studies with isolated perfused rat liver

1. To elucidate the determining factors for elimination pathways of sulfate and glucuronide metabolites of xenobiotics, a single-pass perfusion of 4-methylumbelliferone (4MU) or p-nitrophenol (pNP) was performed with an isolated rat liver preparation.2. Without bovine serum albumin in the perfusion system, clearance calculated based on the unbound concentration in the liver clearly showed that the net efflux clearances (CLeff) of sulfates from the sinusoidal membrane were much higher than those of glucuronides and that the biliary excretion clearances (CLb) of glucuronides were approximately two times larger than those of sulfates.3. The ratios of CLeff to CLb were much higher for sulfates than those for glucuronides. The bile-oriented elimination of glucuronides or sinusoidal efflux-oriented elimination of sulfates was observed even using the perfusate including 3% bovine serum albumin, but the sinusoidal efflux of sulfates was extensively enhanced by bovine serum albumin in the perfusate. The mechanisms behind this stimulatory effect remain to be elucidated.4. For both compounds, CLb of glucuronide was comparable with CLb of sulfate, meaning that CLb is not responsible for the biliary excretion of glucuronides at extensively higher rate than sulfates.5. Higher concentration of glucuronides in the liver, partly caused by much lower CLeff of glucuronides than that of sulfates, is likely responsible for the bile-oriented excretion of glucuronides. The extensive sinusoidal efflux of sulfates, leading to the urine-oriented excretion, is attributed to the substantially higher CLeff than CLb.6. In conclusion, the sinusoidal efflux is an important factor for determining elimination pathways of both sulfates and glucuronides, although further studies are needed to clarify the mechanisms of the sinusoidal efflux.

An investigation of the relationship between the liver and brain using an isolated perfused rat brain preparation

The pathogenesis of portal-systemic encephalopathy (PSE) and hepatic encephalopathy (HE), disorders of the brain attributed to abnormal liver function, are poorly understood. This study was conducted to examine if a fundamental, and possibly exclusive, homeostatic interrelationship exists between the liver and brain that might deteriorate during liver failure to result in the syndrome of PSE and HE. An isolated organ perfusion circuit was devised to accommodate an isolated rat brain and an isolated rat liver preparation perfused concomitantly. The survival time of the brain preparation was measured by the maintenance of the spontaneous electroencephalocorticogram and was extended from a median survival time of 35 min (range 22 to 53 min), when perfused alone, to 210 min (range 172 to 480 min), when perfused simultaneously with a liver. Also, concomitant perfusion with an isolated rat liver reduced perfusate glucose concentrations from 200 mg% to a range between 45 to 60 mg%. These data support our hypothesis that a homeostatic interrelationship exists between the liver and brain that is independent of other metabolic influences; disturbance of this relationship may contribute towards the syndrome of PSE and HE.

Influence of pretreatment with immunosuppressants on O-demethylation of dextromethorphan in isolated perfused rat liver

Changes in the immune system induced by exogenous or endogenous factors may be accompanied with modifications of the activity of the drug metabolising enzymes in the liver. Some immunostimulatory agents are known to suppress the oxidative metabolism mediated by cytochromes P450. Possible effects of substances which suppress the immune responses of the organism have not been fully understood yet. The present study was undertaken to investigate the influence of immunosuppressants cyclophosphamide and dexamethasone on the CYP2D 1-dependent metabolism of dextromethorphan (DEM) in the isolated perfused liver from male rat donors (Wistar albino, 250-310 g). Recirculatory perfusion system was used with Williams' medium E (Sigma Chemicals Co.) as a perfusion medium (120 mL). DEM was administered as a 1 mg bolus into the perfusion solution at the start of each experiment after 20 min preperfusion. Samples of perfusate for HPLC determination of DEM and its O-demethylated metabolite dextrorphan (DOR) were taken at 15 min intervals for 120 min. The results have shown a rapid conversion of DEM to DOR in the isolated rat liver preparations. Pharmacokinetic parameters in the livers from intact rats were as follows: t1/2 DEM = 19.1+/-4.10 min, k(m) = 0.035+/-0.008 min(-1), Cl(m) = 4.21+/-0.97 mL x min(-1), AUC(DOR) = 2160+/-201 microg x min x L(-1). Pretreatment of rats with dexamethasone (4 mg/kg/day, iv, x 3 days) led to a significant increase in the concentration of dextrorphan in the recirculating solution, but it did not substantially change the kinetic constants of DOR formation (km = 0.036+/-0.004 min(-1), Cl(m) = 4.27+/-0.43 mL x min(-1)). Cyclophosphamide (100 mg/kg, ip, 1 dose on day 5 before perfusion) induced nearly twofold increase in the DOR concentrations in perfusate and thus highly significant (p < 0.01) changes of the kinetic parameters characterizing the increased rate of conversion of DEM to DOR (t1/2 DEM = 12.1+/-0.90 min, km = 0.055+/-0.004 min(-1), Cl(m) = 7.09+/-1.37 mL x min(-1), AUC(DOR) = 3602+/-154 microg x min x L(-1)). Considering that both cyclophosphamide and dexamethasone belong to the most widely used immunosuppressive drugs, their potential to promote the CYP2D-mediated metabolism might have a clinical impact in combined therapy of autoimmune or other diseases.

Rat liver transport and biotransformation of a cytostatic complex of bis-cholylglycinate and platinum (II)

Background/Aims: Bile acids have previously been used as shuttles for directing organic drugs to the liver. The aim of this study was to investigate liver transport and biotransformation of a new cytostatic bioinorganic complex (Bamet-H2), that was obtained by binding platinum(II) to two cholylglycinate moieties. Methods: Using rat hepatocytes in primary culture, the kinetics of cholylglycinate, cisplatin and Bamet-H2 uptake were studied. Sodium-dependency of Bamet-H2 uptake was investigated by replacement of 116 mM NaCl by 116 mM choline chloride. Liver biotransformation was investigated by HPLC analysis of bile samples collected from anesthetized rats following intravenous Bamet-H2 administration. Using isolated rat liver preparations, which were perfused with erythrocyte- and albumin-free Krebs-Henseleit solutions for 40 min, measurement of cholylglycinate, cisplatin and Bamet-H2 uptake and bile output was carried out. Interaction between Bamet-H2 and cholylglycinate for liver transport was studied by co-administration of 1 μM Bamet-H2 plus 500 μM cholylglycinate and 1 μM[ 14C]-cholylglycinate plus 500 μM Bamet-H2. Results: Both cholylglycinate and Bamet-H2 uptake by rat hepatocytes followed saturation kinetics. Comparison between the two compounds indicated that the Vmax (22.2 versus 8.5 nmol·5 min −1·mg protein −1), and Kt (365 versus 171 μM) were higher for Bamet-H2 uptake. The efficiency of Bamet-H2 uptake (Vmax/Kt) was significantly reduced (−35%) in the absence of sodium. Cisplatin uptake by rat hepatocytes was approximately 10-fold lower than that for Bamet-H2 at any dose used. Moreover, this was not saturable up to 400 μM cisplatin. Bamet-H2 was not biotransformed during its intrahepatic residence in anesthetized rats. Bamet-H2 uptake and secretion into bile by isolated rat livers exceeded cisplatin but were less than cholylglycinate. Differences between Bamet-H2 and cholylglycinate were more marked for bile output than for liver uptake. Thus, higher drug liver content was found after perfusion with Bamet-H2 than with cholylglycinate or cisplatin. Co-administration of Bamet-H2 and cholylglycinate revealed the existence of partial cross-inhibition in both liver uptake and bile output. Bamet-H2 induced a more profound alteration on cholylglycinate uptake and bile secretion than cholylglycinate on both process for Bamet-H2. Conclusion: These results suggest that in the transfer of Bamet-H2 from the sinusoids to the canaliculi both bile acid and non-bile acid transport systems are involved.

Hemodynamic characteristics of the intrahepatic portal vascular bed over an extended flow range: A study in the isolated perfused rat liver

The relationship between the perfusion pressure (P) and resistance (R) of the intrahepatic portal vascular bed was determined in isolated rat liver preparations perfused with fresh, heparinized rat blood (hematocrit 30%), with rat blood containing a vasodilator agent (sodium nitroprusside, 1 X 10(-3) mol/L), or with 2.5% bovine serum albumin in Krebs-Henseleit buffer (BSA-KH). Pressure-flow curves were constructed over an extended range of portal venous inflow (0 to 70 mL.min-1, corresponding to a flow rate per gram liver wet weight, Q, of approximately 0 to 7 mL.min-1.g-1). Subsequent analysis showed that two mathematical expressions adequately described the data over the full range of flow. Thus, the pressure-flow curve could be represented by (a) the sum of a linear plus a hyperbolic function, i.e., P = Q.R' + Pmax.Q/(Q + Km), where R', Pmax, and Km are constants, or (b) by the simple equation G = C.P, where G is the conductance (Q/P), and C is a conductivity constant. The values of R', Pmax, Km, and C were significantly different under each of the circumstances investigated, but the form of the curve was not altered. Hence, it is proposed that these parameters can be used to describe the fundamental hemodynamic properties of the portal vascular bed of the isolated rat liver. The results are discussed in terms of the microvascular recruitment and distensible resistance vessel models of the hepatic microcirculation.

Hemodynamic characteristics of the intrahepatic portal vascular bed over an extended flow range: A study in the isolated perfused rat liver

The relationship between the perfusion pressure (P) and resistance (R) of the intrahepatic portal vascular bed was determined in isolated rat liver preparations perfused with fresh, heparinized rat blood (hematocrit 30%), with rat blood containing a vasodilator agent (sodium nitroprusside, 1 × 10 −3 mol/L), or with 2.5% bovine serum albumin in Krebs-Henseleit buffer (BSA-KH). Pressure-flow curves were constructed over an extended range of portal venous inflow (0 to 70 mL·min −1, corresponding to a flow rate per gram liver wet weight, Q, of approximately 0 to 7 mL·min −1·g −1). Subsequent analysis showed that two mathematical expressions adequately described the data over the full range of flow. Thus, the pressure-flow curve could be represented by (a) the sum of a linear plus a hyperbolic function, i.e., P = Q·R′ + Pmax·Q/(Q + Km), where R′, Pmax, and Km are constants, or (b) by the simple equation G = C·P, where G is the conductance (Q/P), and C is a conductivity constant. The values of R′, Pmax, Km, and C were significantly different under each of the circumstances investigated, but the form of the curve was not altered. Hence, it is proposed that these parameters can be used to describe the fundamental hemodynamic properties of the portal vascular bed of the isolated rat liver. The results are discussed in terms of the microvascular recruitment and distensible resistance vessel models of the hepatic microcirculation.

Contribution by the glycogen pool and adenosine 3',5'-monophosphate release to the evanescent effect of glucagon on hepatic glucose production in vitro.

To elucidate in vitro the transience of glucagon-induced hepatic glucose release, the effects of glucagon on hepatic glucose production and cAMP release were evaluated in the isolated rat liver preparation perfused by a nonrecirculating system. Glucagon was added to the infusate in stepwise increasing concentrations at 0, 60, and 100 min to give final concentrations of 2.5 X 10(-11), 10(-9), and 5 X 10(-8) M, respectively. Glucagon at 2.5 X 10(-11) M caused cAMP release [basal (mean +/- SD), 11.2 +/- 3.0 pmol/(min X 100 g BW)] to rise rapidly and plateau at 23.3 +/- 7.0 pmol/(min X 100 g BW), whereas hepatic glucose production [basal, 3.7 +/- 1.6 mumol/(min X 100 g BW)] increased only transiently to a maximum of 15.3 +/- 3.1 mumol/(min X 100 g BW) and fell thereafter. The enhanced cAMP release during the consecutive glucagon infusion was accompanied by a transient rise in hepatic glucose production during the second, but not during a third, glucagon infusion. When 3-isobutyl-1-methylxanthine, a potent phosphodiesterase inhibitor, was added to the perfusion medium (0.5 mM), the cAMP response to 2.5 X 10(-11) M glucagon was enhanced [247 +/- 124 pmol/(min X 100 g BW)] as was hepatic glucose production (+ 21%; P less than 0.05). Further augmentation of the glucagon concentration was followed by an increase in hepatic cAMP, but not glucose, release. When glucagon infusion (2.5 X 10(-11) M) was repeated with a glucagon-free period of 30 min in between, no stimulation of cAMP and consecutive glucose release was found during the second period. However, when the second glucagon dose was increased to 10(-9) M, glucose and cAMP release were again stimulated to the same extent as in experiments with no glucagon-free period in between. We conclude that the size of the glycogen pool and the cAMP concentration directly modulate hepatic glucose production and are responsible for evanescent glucagon action. This mechanism can be described by computer simulation.

The simultaneous measurement of thermal conductivity, thermal diffusivity, and perfusion in small volumes of tissue.

An improved technique is presented for the "in-vivo" determination of thermal conductivity, thermal diffusivity, and perfusion using a self-heated spherical thermistor probe. In the presence of flow, solution of the time-dependent, probe-tissue coupled thermal model allows the measurement of "effective" thermal conductivity and "effective" thermal diffusivity, which represent the thermal properties of the perfused tissue. Perfusion can be quantified from both "effective" thermal properties. In the presence of flow, it has been shown that the transient power responses does not follow t-1/2 as has been previously assumed. An isolated rat liver preparation has been developed validate the measurement technique. Radioactive microspheres are used to determine the true perfusion from the total collected hepatic vein flow. Experimental data demonstrates the ability to quantify perfusion in small volumes of tissue.

Uptake of phospholipid-depleted chylomicrons by the perfused rat liver.

1. Rat lymph chylomicrons were depleted of their surface phospholipids by treatment with pure phospholipase A2 from Crotalus adamanteus venom. 2. About 80% of the phospholipids could be removed from the chylomicrons without any apparent effect on their size, neutral lipid composition or qualitative profile of their tetramethylurea-soluble apoproteins. 3. Phospholipid-depleted chylomicrons were rapidly taken up whole by liver cells when perfused through isolated rat liver preparations. The rate of uptake was dependent on the extent of phospholipid depletion and reached a maximum (4-6.5-fold greater than control chylomicrons) when 80% of the phospholipids had been removed. 4. It is speculated that the hepatic uptake of phospholipid-depleted chylomicrons occurs by a mechanism to that of chylomicron-remnants uptake.

PGE 1 metabolism by the perfused rat liver

The hepatic and biliary metabolites of PGE 1 have been isolated and identified after infusions of PGE 1 into isolated rat liver preparations. The results demonstrate that in general PGE 1 undergoes metabolism similar to that of PGE 2 in the rat and reveals the possibility of a selective PG metabolite transport system across the biliary canalicular membrane.

The effects of glucose and of potassium ions on the interconversion of the two forms of glycogen phosphorylase and of glycogen synthetase in isolated rat liver preparations.

In the isolated perfused rat liver, increasing glucose concentration from 5.5 to 55 mm in the perfusion medium caused a sequential inactivation of glycogen phosphorylase and activation of glycogen synthetase. The latter change was preceded by a lag period which corresponded to the time required to inactivate the major part of the phosphorylase. 2. The same sequence of events was observed in isolated rat hepatocytes incubated at 37C. In this preparation, the rate of phosphorylase inactivation was greatly increased by increasing the concentration of glucose and/or of K+ ions in the external medium. The same agents also caused the activation of glycogen synthetase, but this effect was secondary to the inactivation of phosphorylase. 3. In both types of preparations, the rate of synthetase activation was modulated by the residual amount of phosphorylase a that remained after the initial phase of rapid inactivation and was independent of glucose concentration. 4. In isolated hepatocytes, the rate of conversion of glucose into glycogen was propotional to the activity of synthetase a in the preparation. This conversion was preceded by a lag period which could be shortened by increasing either glucose or K+ concentration in the medium. The incorporation of labelled glucose into glycogen was simultaneous with a glycogenolytic process which could not be attributed to the activity of phosphorylase a.

A compact perfusion system for studies on the control of metabolic processes in isolated organs from small animals.

A compact apparatus for perfusion of isolated organs from small animals is described. The system is based on a module principle making it useable both in low and high pressure perfusions. 40-80 ml of heparinized, diluted rat blood used as liver-perfusion medium, is recirculated in the system, which is temperature controlled by means of a water jacket. A new type of capillary membrane oxygenator is used for oxygenation. The system permits rapid measurements of perfusate flow, vascular resistance, bile production (when the isolated liver is used), urinary production (when the isolated kidney is used), gas tensions, oxygen consumption, pH and perfusion pressure. Descriptions of the techniques used for operation and perfusion of isolated livers and kidneys from rats are given. By the generally accepted criteria of viability the isolated rat liver remains almost normal for the test period of 3 h. Liver functions were normal as judged by formation of glucose, respiratory rate, urea and bile production and gross morphology. There was very little leakage of liver enzymes into the perfusate. The transformation and elimination of labelled corticosterone were studied to test the steroid metabolizing activity of the isolated rat liver preparation.

Effect of leucine on carbohydrate metabolism of the liver.

The respective effects of certain amino acids on carbohydrate metabolism of the liver have been studied in isolated rat liver preparations. It has been shown that - unlike the other amino acids investigated - leucine generally enhances glucose release of the liver while increasing its glycogen content. Given simultaneously with insulin of a concentration ineffective in itself, leucine increased the effect of insulin on liver metabolism. The significance of the observation in the reduction of blood sugar upon the effect of leucine is discussed.

Inactivation of penicillins by the isolated rat liver.

Abstract Prolonged ampicillin blood levels have been attributed to recycling through the biliary system. The present study was undertaken to make specific measurements of the role of inactivation and biliary excretion of penicillin G and ampicillin by the liver with an isolated rat liver preparation. Varying amounts of the drugs were circulated through the rat liver, antibiotic concentrations were measured, and inactivation rates were calculated. Radioactive penicillins were also administered to determine the fate of degradation products. Ampicillin was inactivated only half as rapidly as penicillin G. Small, but equal amounts of each drug were excreted in the bile. Thus, the higher and more prolonged blood levels of ampicillin appear to be due to a lower rate of inactivation by the liver rather than to recycling through the biliary system.

LIVER INJURY IN ISOLATED PERFUSED RAT LIVER PREPARATION EXPOSED TO CHLOROFORM

Exposure of the isolated rat liver preparation to CHCl 3 at concentrations of 0.016–0.056 mg/l. O 2 /CO 2 results in decreased bile flow, increased glucose concentrations in the perfusate, increased tissue sodium space and early histological changes similar to those seen in vivo . These changes are associated with slight vasodilatation and increased rates of blood flow through the liver. Withdrawal of CHCl 3 does not result in reversal of any of the above changes except for the vasodilatation over a period of 3 hours after termination of the chloroform exposure. The effect of differences in CHCl 3 dosage is presented. The data are interpreted as a ) confirming the view that a biochemical lesion rather than circulatory events initiates the chloroform injury to the liver and b ) underscoring the vulnerability of the tissue during the protracted period of recovery from the damage inflicted by this agent.

Liver injury in isolated perfused rat liver preparation exposed to chloroform

Exposure of the isolated rat liver preparation to CHCl3 at concentrations of 0.016–0.056 mg/l. O2/CO2 results in decreased bile flow, increased glucose concentrations in the perfusate, increased tissue sodium space and early histological changes similar to those seen in vivo. These changes are associated with slight vasodilatation and increased rates of blood flow through the liver. Withdrawal of CHCl3 does not result in reversal of any of the above changes except for the vasodilatation over a period of 3 hours after termination of the chloroform exposure. The effect of differences in CHCl3 dosage is presented. The data are interpreted as a) confirming the view that a biochemical lesion rather than circulatory events initiates the chloroform injury to the liver and b) underscoring the vulnerability of the tissue during the protracted period of recovery from the damage inflicted by this agent.

Liver function in regenerating rat liver. CrPO4 colloid uptake and bile flow

Functional changes in the regenerating hepatic tissue as indicated by CrPO4 colloid extraction and bile secretion by the isolated rat liver preparation were studied. Following partial hepatectomy, the liver weight/body weight ratio of the operated rats is significantly lower than the nonoperated controls. This is attributed to only a 80–85% restoration of the residual hepatic tissue. A transient increase in bile secretion/ unit weight of liver by 50% above the preoperative level occurs within 48 hours after partial resection. The uptake of radioactive chromic phosphate colloid indicates an increase in Kupffer cell activity (phagocytic functions) to values 200% above control within 48 hours. This function, however, is sustained and remains elevated for at least 90–100 days postoperatively. Histological evidence indicates that the increased Kupffer cell activity is probably due to an increase in cell population of phagocytic cells. Theoretical considerations for these changes in specific function of regenerating hepatic tissue and their relation to normal resting liver are discussed.

Changes in liver function and structure due to experimental passive congestion under controlled hepatic vein pressures.

The effect of various hepatic vein pressures on hepatic hemodynamics, on fluid shifts within the liver, and on certain indices of liver function were studied in the isolated rat liver preparation. In general, venous pressures are scarcely perceived by the hepatic parenchyma unless they exceed the hydrostatic pressures acting at the liver surface, more particularly at the hilum of the liver. Venous pressures exceeding this value appear to distend the liver vasculature so that it acts like a system of rigid conduits; there is great expansion of the sodium space and of the colloid distribution spaces, and some evidence of partial sequestration of blood in the congested liver. All these changes impair oxygen supply to the tissue. To a lesser extent they interfere with the transfer of other substances from blood to parenchyma, and place considerable stress upon the mechanical framework of the liver. The resulting strain in turn tends to limit the extent of intrahepatic changes in passive congestion, and possibly the rate of fluid leakage from blood via parenchyma to transudate at the liver surface.

Hemodynamics of the vascular tree of the isolated rat liver preparation.

The hemodynamic properties of the hepatic vascular bed of the isolated rat liver preparation have been studied by the use of flow-pressure diagrams in experiments involving as a perfusate either whole blood or cell-free rat plasma under an oxygen partial pressure of 2.5 atm. Either perfusion medium yields a nonlinear flow-pressure diagram of sigmoid shape approaching a straight line through the origin at perfusion pressures slightly above physiological ones. Data are also reported concerning change of liver weight with perfusion pressure of the isolated organ. These, together with previously reported results of radiocolloid uptake studies, are incorporated into a representation of the hepatic vascular bed in terms of a population of parallel vascular channels having critical opening pressures distributed between 7 and 13 cm of plasma. The distribution function is described and its bearing upon liver function in relation to liver blood flow is briefly discussed.

Circulatory pathways in the rat liver as revealed by P32 chromic phosphate colloid uptake in the isolated perfused liver preparation.

P32-labeled chromic phosphate colloid disappears from the circulation of the isolated rat liver preparation according to a single exponential term of time. A small nonextracted contaminant, less than 3% of the total activity is also detected. At comparable blood flow rates the colloid is extracted about as completely by the isolated liver preparation as by the liver in situ in the intact animal. The efficiency with which chromic phosphate colloid is removed from perfusate passing through the isolated rat liver decreases with increasing perfusion rate. If whole blood is used as a perfusate, the efficiency of colloid extraction is almost twice as high as it is if rat blood plasma is employed, even if adequate oxygenation of the tissue is assured by high oxygen partial pressures in the latter series. A theoretical treatment of these results is given in terms of first order reaction kinetics. Agreement of experimental results with this theory at perfusion rates greater than 2 cc/gm/min. indicates that the rate of chromic phosphate colloid extraction is a function of plasma concentration of the colloid, and that the extraction efficiency for a given perfusate varies as a function of the mean transit time of perfusate through the liver. Deviations from the predictions of the theory occur at low perfusion rates, and are discussed in the light of the above concepts. A decrease of the ratio of transit time to perfusion rate under these conditions suggests a decrease in the number of channels open to blood flow at low perfusion pressures.