Uridine catabolism by the isolated perfused rat liver

A new approach in the treatment of gastrointestinal tumors with 5-fluorouracil involves the infusion of high doses of uridine to improve the chemotherapeutic efficiency of the former. High amounts of uracil formed from uridine can interfere with the hepatic catabolism of 5-fluorouracil and thus increase its bioavailability and toxicity. In our study, we analysed the metabolite pattern of uridine in the effluent of isolated perfused rat livers in relation to portal uridine levels. The livers were perfused hemoglobin-free without recirculation at a constant flow. In the perfusate, uridine was changed from 0.5 to 100 mumol/l. The complete degradation of [2-14C]uridine and [2-14C]uracil was monitored via the release of labeled CO2. Radioactive catabolites of uridine including uracil and the sum of dihydrouracil and beta-ureidopropionate were separated by high-performance liquid chromatography and counted using a radioactivity flow monitor. Portal uridine concentrations were increased from 0.5 to 100 mumol/l and were accompanied by a rise in the relative amount of non-metabolized uridine in the effluent from 13 to 78%. At uridine concentrations above 50 mumol/l, there was a constant release of uracil into the effluent, indicating saturation of uridine phosphorolysis or transport. The amount of 14CO2 formed by the liver reflecting complete uridine breakdown was higher than any other uridine metabolite when uridine concentration varied from 0.5 to 15 mumol/l. Saturation of 14CO2 formation was achieved at a uridine concentration of 25 mumol/l. Higher peak values of 14CO2 release were observed after direct infusion of equivalent amounts of uracil into the portal vein.(ABSTRACT TRUNCATED AT 250 WORDS)     

Synthesis of fibronectin by the isolated perfused rat liver

The major site(s) of synthesis of plasma fibronectin is unknown. Using the isolated perfused rat liver and anti-rat fibronectin antiserum, we have measured net hepatic synthesis of fibronectin during 10-hr perfusion periods. We calculated the circulating plasma fibronectin pool of a 195-g rat with body surface area of 300 sq cm to be 3.3 mg: net hepatic synthesis of 3.75 +/- 1.0 mg/300 sq cm body surface area of the rat liver donor was seen in liver perfusions of 10 hr. When the hormones cortisol and insulin were added to the liver perfusate, net synthesis of 5.05 +/- 0.82 mg/300 sq cm was seen. Synthesis of two known acute phase reactant proteins, fibrinogen and alpha-2 acute phase globulin, was also substantially greater in the presence of cortisol and insulin. Plasma fibronectin levels measured in intact rats that had abscess produced by subcutaneous turpentine injection were 0.57 +/- 0;05 mg/ml compared to 0.38 +/- 0.02 mg/ml in normal control animals. Our studies indicate that hepatic synthesis contributes substantially to the plasma fibronectin pool and show that such synthesis is enhanced by the hormones cortisol and insulin. In the intact rat, inflammation was associated with elevated levels of plasma fibronectin.     

Effect of cirrhosis on sulphation by the isolated perfused rat liver

BACKGROUND/AIMS: There is evidence to suggest that not all pathways of drug metabolism are similarly affected in cirrhosis. The effect of cirrhosis on drug oxidation and glucuronidation has been extensively investigated but little is known of the effect of cirrhosis on drug sulphation. The aim of this study was to investigate the effect of cirrhosis on sulphation. METHODS: We investigated the effect of cirrhosis on p-nitrophenol sulphation and compared this with the effect of cirrhosis on p-nitrophenol glucuronidation as well as on d-propranolol oxidation simultaneously in the single-pass isolated perfused rat liver. The perfusate contained added inorganic sulphate to maximise production of p-nitrophenol sulphate. RESULTS: About 77% and 59% of p-nitrophenol was eliminated as the sulphate conjugate by the healthy (n=6) and cirrhotic (n=7) livers, respectively. Mean total p-nitrophenol clearance was decreased in cirrhosis (healthy: 18.5+/-0.2 vs. cirrhotic 15.3+/-4.0 ml/min; p<0.05). The decrease in total clearance of p-nitrophenol was due solely to the decrease in sulphate formation clearance, which was significantly decreased (healthy: 14.1+/-1.9 vs. cirrhotic: 9.27+/-3.33 ml/min; p<0.05). Mean glucuronide formation clearance (healthy: 5.11+/-0.94 vs cirrhotic: 5.79+/-0.85 ml/ min; p>0.05) was not significantly altered. Mean total propranolol clearance was decreased in cirrhosis (healthy: 19.9+/-0.1 vs. cirrhotics: 18.0+/-1.5 ml/min; p<0.05). CONCLUSIONS: We have shown that in cirrhosis there is significant impairment of drug oxidation and sulphation, whilst glucuronidation is spared. The decreased sulphation of p-nitrophenol was most likely due to a decrease in phenol sulphotransferase and/or decrease in cofactor synthesis.     

Decreased uptake of taurocholate and ouabain by hepatocytes isolated from cirrhotic rat liver

To differentiate between the "intact" and "sick" cell hypothesis explaining decreased clearance of endo- and xenobiotics, we measured uptake of taurocholate and ouabain in hepatocytes isolated from cirrhotic rat liver. Cirrhosis was induced by chronic exposure of male Sprague-Dawley rats to phenobarbital and carbon tetrachloride. Uptake of [14C]taurocholate and [3H]ouabain was measured by a rapid filtration technique. Hepatocytes from cirrhotic liver were as viable as control hepatocytes--as judged by trypan blue exclusion and lactate dehydrogenase release--but consumed 28% less oxygen. Vmax of both taurocholate (3.16 +/- 0.95 vs. 0.40 +/- 0.35 nmoles X min-1 X 10(6) cells-1; p less than 0.001) and ouabain (2.16 +/- 0.78 vs. 0.83 +/- 0.26 nmoles X min-1 X 10(6) cells-1; p less than 0.005) was significantly reduced. These results are compatible with the "sick" cell hypothesis.     

Substrate effects on mitochondrial function and tissue lipids in low-flow hypoxia of isolated perfused rat hearts

Summary A possible causal relationship between tissue FFA contents and the depression in mitochondrial oxidative phosphorylation in myocardial ischaemia has been suggested. To test this hypothesis, the effects of different substrates added to the perfusates of hypoxic, low-flow perfused hearts were examined on oxidative phosphorylation catalysed by mitochondria isolated from such tissue. In an additional series of experiments tissue neutral glyceride and FFA levels were analysed and correlated with changes in mitochondrial function. Mitochondria isolated from hearts with a high tissue FFA content exhibited the lowest ADP/O ratios, RCI and QO₂ values. On the other hand, mitochondria isolated from hearts with reduced FFA contents, performed significantly better with respect to these parameters of mitochondrial function studied.

---

Substrateffekte auf Mitochondrienfunktion und Gewebelipide des isolierten Rattenherzens bei Hypoxie infolge Mangelperfusion

Zusammenfassung Aufgrund früherer Untersuchungen wurde eine kausale Beziehung zwischen dem Gehalt des Gewebes an freien Fettsäuren und der Depression der oxidativen Phosphorylierung in den Mitochondrien bei Myokadischämie vermutet. Um diese Hypothese zu testen, wurden dem Perfusat hypoxischer, mangelperfundierter Herzen verschiedene Substrate zugefügt und deren Einfluß auf die oxidative Phosphorylierung der aus solchen Geweben isolierten Mitochondrien untersucht. In weiteren Experimenten wurde der Gewebsspiegel der neutralen Glyzeride und freien Fettsäuren analysiert und mit Änderungen der Mitochondrienfunktion korreliert. Mitochondrien, die aus Herzen mit einem hohen Gehalt an freien Fettsäuren isoliert worden waren, zeigten die geringsten Werte für das Verhältnis ADP/0, sowie für den respiratorischen Kontrollindex (RCI) und die mitochondriale Sauerstoffaufnahme (QO₂). Andererseits war die Funktion-gemessen an diesen Parametern-eindutig besser bie Mitochondrien, die von Herzen mit reduziertem Gehalt an freinen Fettsäuren gewonnen wurden.

---

---

With 2 figures and 3 tables     

Phagocytosis activity of the isolated perfused rat liver

The reticulohistiocytary system was tested with colloidal carbon. Isolated livers were perfused at 20 degrees C, 28 degrees C, 37 degrees C and 45 degrees C with a hemoglobin and protein free medium. It was shown that the perfusion at 28 degrees C revealed a significantly better phagocytosis as at temperatures of 37 degrees C and 45 degrees C. The control parameters of the liver function got, gpt lactate-pyruvate, beta-hydroxybutyrate and acetoacetate displayed the best results at 28 degrees C.     

Uptake and catabolism of gamma-aminobutyric acid by the isolated perfused rat liver

Serum concentrations of gamma-aminobutyric acid (GABA) are increased in liver failure, possibly because of decreased hepatic GABA catabolism. To study in detail the role of the liver in GABA metabolism, uptake and catabolism of GABA by isolated perfused liver from normal rats and rats with galactosamine- or carbon tetrachloride-induced liver failure were measured. Hepatic GABA uptake was almost complete at GABA concentrations of up to 10 microM and approached saturation at a concentration of 50 microM. The apparent affinity of hepatic GABA uptake was 38 microM and the apparent maximal velocity was 158 nmol/g.min. Hepatic GABA uptake was sodium-dependent. gamma-Aminobutyric acid taken up by the liver was rapidly catabolized as measured by 14CO2 formation from [U-14C]GABA. Aminooxyacetic acid, a GABA transaminase inhibitor, completely and irreversibly inhibited hepatic GABA catabolism and thereby also inhibited hepatic GABA uptake. Although uptake of GABA by livers of carbon tetrachloride- or galactosamine-treated rats was decreased (apparent maximal velocity, 103 and 98 nmol/g.min, respectively), at physiologic GABA concentrations in the perfusate GABA uptake and catabolism was not different from that of untreated controls. The observed impairment of hepatic GABA uptake or catabolism by the diseased liver would be expected to contribute to increased GABA levels in peripheral blood plasma in liver failure. However, the magnitude of the observed impairment would be insufficient to account for a 10-fold increase in such levels.     

Early midzonal cell death during low-flow hypoxia in the isolated, perfused rat liver: protection by allopurinol

Trypan blue uptake and lactate dehydrogenase release were measured as indices of irreversible cell damage in isolated, perfused rat livers during low-flow hypoxia. In livers from fasted rats perfused in the anterograde direction, trypan blue uptake took place beginning at about 45 min of hypoxia. Cells which took up trypan blue first were located in narrow bands at the border between anoxic pericentral areas and normoxic periportal regions of the liver lobule. After longer periods of hypoxia, trypan blue uptake progressed towards the central vein until after 120 min virtually all cells in the pericentral regions were stained. Under these conditions, cells in periportal regions were spared. In perfusions in the retrograde direction, cell death began in midzonal regions and spread towards the portal vein. Release of lactate dehydrogenase into the effluent paralleled trypan blue uptake, beginning at about 40 min of low-flow hypoxia and peaking at 80 min. In contrast to livers from fasted rats, trypan blue was not taken up, and lactate dehydrogenase was not released in livers from fed rats exposed to low-flow hypoxia for as long as 120 min. To test the hypothesis that xanthine oxidase-mediated oxygen-free radical formation was involved in cell injury at the border between anoxic and normoxic regions (anoxic edge), allopurinol, an inhibitor of xanthine oxidase, was studied. Allopurinol (0.2 to 5 mM) delayed the release of lactate dehydrogenase during low-flow hypoxia in a dose-dependent fashion (e.g., 5 mM allopurinol delayed hypoxia-induced lactate dehydrogenase release by about 30 min).(ABSTRACT TRUNCATED AT 250 WORDS)     

The elimination of aged glycoproteins by the isolated perfused rat liver

The isolated livers of Sprague-Dawley rats aged ten months were perfused with nine different glycoproteins (coeruloplasmine, haptoglobin, transferrin, alpha2-mocroglobulin, apolipoprotein A, plasminogen, acid alpha1-glycoprotein, alpha1-antitrypsin, beta2-glycoprotein), which were suspended in a basic medium free of hemoglobin and other proteins. Besides a control group two other groups were formed to give a model for aging: one group of glycoproteins was desialysed, another was desilysed and incubated with immunoglobulins before the perfusion. In spite of the application of two procedures which are supposed to produce a model of old glycoproteins, neither treatment resulted in the recognition, binding and elimination of the glycoproteins from the perfusion medium.     

Hyperglycemia-induced cholestasis in the isolated perfused rat liver

In a previous report we showed that cholestasis in diabetic rats is due in part to hyperglycemia. To gain information about the mechanism responsible for this phenomenon, bile flow was studied in isolated perfused rat livers. The perfusion media were modified erythrocyte-free Krebs-Henseleit solutions. Under these experimental conditions, no cholestasis was observed in isolated rat livers obtained from rats treated with streptozotocin (6 mg/100 gm body wt) 6 days before the experiments. We then proceeded to use normal animals. The composition of the perfusion media was modified to maintain the osmolality even after increasing D-glucose concentrations from 0 to 35 mmol/L. Bile flow was not affected with doses up to 15 mmol/L D-glucose. Beyond a threshold value for plasma D-glucose concentrations between 15 and 20 mmol/L, cholestasis was observed. Using D-glucose analogs such as L-glucose and 3-O-methyl-D-glucose, bile flow was also reduced (by 0.54 and 0.53 microliters/min/gm liver, respectively). Isosmotic sucrose-containing perfusion media were also observed to impair bile flow (by 0.66 microliters/min/gm liver). However, i-erythritol and D-mannitol failed to inhibit bile formation. The study of bile/plasma concentration ratios determined using tracer amounts of radioactive sugars indicated that this value was much lower for cholestatic sucrose (0.11) and L-glucose (0.31) than for noncholestatic i-erythritol (0.99) and D-mannitol (0.98). Cholestasis was partly reversed after induction by 35 mmol/L D-glucose if perfusion media were replaced by sugar-free ones, but also by media containing 25 mmol/L D-glucose. Insulin given during the perfusion with sugar-free media was observed to have no effect on bile flow.(ABSTRACT TRUNCATED AT 250 WORDS)     

Uptake of free choline by isolated perfused rat liver.

The uptake of free choline by isolated perfused rat liver was characterized. A saturable uptake mechanism [Ka = 0.17 +/- 0.07 mM (SD); Vmax = 0.84 +/- 0.16 mumol/min X g dry weight] and a nonsaturable mechanism (through which uptake is proportional to choline concentration in the perfusate) were identified. Most of the choline transported into hepatocytes was converted to betaine, phosphorylcholine, or lecithin. Free choline also accumulated within the intracellular space, suggesting that choline oxidase activity does not always limit choline's uptake by the liver.     

Production and release of plasminogen by isolated perfused rat liver.

In view of conflicting evidence for a major hepatic role in the synthesis of circulating plasminogen, the precursor of the fibrinolytic enzyme plasmin, we carried out the present study with a sensitive assay in order to measure the accumulation of small quantities of plasminogen in a recycling rat liver perfusion system. We have purified plasminogen from Sprague-Dawley rat plasma and have raised a monospecific antiserum against it in rabbits. Isolated rat liver perfusions were performed with an oxygenated recycling perfusate consisting of a perfluorotributylamine/Pluronic F-68 emulsion (Fluosol 43) free of plasma proteins and blood cells. The system was shown to be capable of synthesizing albumin and transferrin. The cumulative appearance of plasminogen in the perfusate was measured by a sensitive, specific radioimmunoassay. Plasminogen concentration increased progressively during the first 2 hr of perfusion; the observed average net synthesis in five separate experiments was approximately 35 microgram/hr per 100 g of body weight. Exposure of the perfused liver to 18 microM cycloheximide inhibited additional increase in the titer of plasminogen. Evidence for de novo synthesis was provided by the incorporation of 14C-labeled leucine into specific immunoprecipitates of plasminogen and the inhibition of this incorporation by cycloheximide. Analysis of the immunoprecipitates by sodium dodecyl sulfate/polyacrylamide gel electrophoresis revealed a single peak of radioactivity corresponding to Mr of 82,000. These data indicate that the liver is a major site of plasminogen production.     

Protective effects of regulatory amino acids on ischemia-reperfusion injury in the isolated perfused rat liver

OBJECTIVE: Some amino acids (AAs) display potent regulatory activities on cell metabolism, including via anti-oxidative defences. The aim of this study was to evaluate the protective effect of these AAs on warm ischaemia-reperfusion (I/R) injury in the isolated perfused rat liver. MATERIAL AND METHODS: Livers from fasted male Sprague-Dawley rats were isolated and perfused without (control group) or with (AP group) a mixture of regulatory AAs (glutamine, histidine, leucine, methionine, proline, phenylalanine, tryptophan and alanine). After 45 min of perfusion, warm ischaemia was induced for 45 min by clamping the portal vein catheter; thereafter, reperfusion was performed for 30 min. RESULTS: TNF-alpha production was significantly lower in the AP group during reperfusion (Control: 39+/-7 versus AP: 16+/-2 pg min-1 g-1, p<0.05), and lactate dehydrogenase (LDH) release decreased significantly during the last 15 min of reperfusion (Control: 0.13+/-0.03 versus AP: 0.04+/-0.02 IU min-1 g-1, p<0.05), despite similar levels of oxidative stress. The addition of regulatory AAs was not associated with variations in portal flow, bile flow, hepatic glucose or urea metabolism. However, significant changes in intrahepatic glutamine (Control: 1.4+/-0.2 versus AP: 2.6+/-0.5 micromol g-1, p < 0.05) together with higher glutamate release in the AP group (Control: 10.2+/-5.4 versus AP: 42.6+/-10.9 nmol min-1 g-1, p < 0.05) indicated modifications in nitrogen metabolism. CONCLUSIONS: Taken together, the lower TNF-alpha production, suggesting decreased inflammatory response, the decrease in LDH release in the AP group, demonstrating a better preservation of liver viability, and the increase in hepatic glutamine indicate that AAs play an important role in the liver's response to I/R.     

Protective effects of regulatory amino acids on ischaemia-reperfusion injury in the isolated perfused rat liver

Objective. Some amino acids (AAs) display potent regulatory activities on cell metabolism, including via anti-oxidative defences. The aim of this study was to evaluate the protective effect of these AAs on warm ischaemia-reperfusion (I/R) injury in the isolated perfused rat liver.

Material and methods. Livers from fasted male Sprague-Dawley rats were isolated and perfused without (control group) or with (AP group) a mixture of regulatory AAs (glutamine, histidine, leucine, methionine, proline, phenylalanine, tryptophan and alanine). After 45 min of perfusion, warm ischaemia was induced for 45 min by clamping the portal vein catheter; thereafter, reperfusion was performed for 30 min.

Results. TNF-α production was significantly lower in the AP group during reperfusion (Control: 39±7 versus AP: 16±2 pg min^?1 g^?1, p<0.05), and lactate dehydrogenase (LDH) release decreased significantly during the last 15 min of reperfusion (Control: 0.13±0.03 versus AP: 0.04±0.02 IU min^?1 g^?1, p<0.05), despite similar levels of oxidative stress. The addition of regulatory AAs was not associated with variations in portal flow, bile flow, hepatic glucose or urea metabolism. However, significant changes in intrahepatic glutamine (Control: 1.4±0.2 versus AP: 2.6±0.5 µmol g^?1, p<0.05) together with higher glutamate release in the AP group (Control: 10.2±5.4 versus AP: 42.6±10.9 nmol min^?1 g^?1, p<0.05) indicated modifications in nitrogen metabolism.

Conclusions. Taken together, the lower TNF-α production, suggesting decreased inflammatory response, the decrease in LDH release in the AP group, demonstrating a better preservation of liver viability, and the increase in hepatic glutamine indicate that AAs play an important role in the liver's response to I/R.     

Effect of nafenopin on the uptake of bilirubin and sulfobromophthalein by isolated perfused rat liver

Hepatic uptake of bilirubin and sulfobromophthalein has kinetic characteristics suggesting facilitated diffusion. Because these compounds demonstrate mutual competition for uptake, a shared uptake mechanism has been presumed. Previous studies in isolated perfused regenerating liver revealed depressed uptake of bilirubin, sulfobromophthalein, and asialoorosomucoid, a desialylated glycoprotein which enters hepatocytes by receptor-mediated endocytosis. This study was designed to determine whether or not depressed transport seen in liver regeneration occurs in other states of hepatocellular proliferation. Rats were pretreated with nafenopin (200 mg/kg . day x 2), a drug that causes rapid hepatocellular proliferation similar to that seen in regeneration. Twenty-four hours after nafenopin treatment, liver weight increased by 40%. Influx, efflux, and sequestration rate constants in isolated perfused liver were quantitated by a computer fit to the model of Goresky. Results 1 day after nafenopin treatment revealed no change in transport parameters for bilirubin and asialoorosomucoid, but 55% and 49% reductions in influx of sulfobromophthalein and conjugated bilirubin, respectively. These studies suggest that hepatocellular proliferation alone is not responsible for the transport alterations seen during liver regeneration. Nafenopin effectively unmasks differences in uptake of bilirubin and other more water soluble organic anions such as sulfobromophthalein and conjugated bilirubin, suggesting that their uptake mechanisms are partially independent.     

The disposition of 6-deoxyacyclovir, a xanthine oxidase-activated prodrug of acyclovir, in the isolated perfused rat liver

The antiviral drug, acyclovir, has been used in the treatment of chronic type B hepatitis. High serum concentrations of acyclovir are required to achieve inhibition of hepatitis B viral replication. Because only 15 to 20% of an oral dose is absorbed, it is necessary to administer acyclovir by intravenous infusion. 6-Deoxyacyclovir, an analog of acyclovir, is well absorbed when given orally, and is converted to acyclovir by xanthine oxidase which is present in the gut and liver. This study has examined the hepatic disposition of 6-deoxyacyclovir in a 100 ml recirculating (12 ml per min) perfused rat liver system. Following administration of a bolus dose of 5 mumoles 6-deoxyacyclovir to the reservoir, perfusate concentrations of 6-deoxyacyclovir declined monoexponentially, as the metabolite acyclovir appeared in the perfusate. Addition of the xanthine oxidase inhibitor allopurinol (5 mg) to the perfusate reservoir prior to the administration of 6-deoxyacyclovir resulted in impaired hepatic metabolism of 6-deoxyacyclovir, as demonstrated by a 47% reduction in systemic clearance rate (4.5 +/- 0.4 to 2.4 +/- 0.9 ml per min; p less than 0.05) (mean +/- S.E., n = 6) and a 1.8-fold increase in terminal elimination half-life of 6-deoxyacyclovir (23.5 +/- 2.7 to 42.7 +/- 4.1 min; p less than 0.05), accompanied by a 30% reduction in appearance of acyclovir. The efficient hepatic conversion of 6-deoxyacyclovir to the active antiviral drug, acyclovir, provides a rationale for trials of oral 6-deoxyacyclovir in the treatment of chronic type B hepatitis.