Hemoglobin-free Perfused Rat Liver Research Articles

Low Doses of Tumour Necrosis Factor α and Interleukin 1β Diminish Hepatic Gluconeogenesis from Alanine in vivo

Previous reports have attributed a stimulating action on hepatic gluconeogenesis to tumour necrosis factor alpha (TNFalpha) administered to rats at high doses (250 mug/kg). However, in adjuvant-induced arthritic rats, which present TNFalpha and other interleukins in the circulation, hepatic gluconeogenesis is diminished. The same occurs in some types of experimental cancer models as, for example, rats bearing the Walker-256 tumour. The present work represents an attempt of reproducing in rats gluconeogenesis inhibition by interleukins using low instead of high doses of both TNFalpha and interleukin 1beta (IL1beta). TNFalpha and IL1beta at doses of up to 10 mug/kg were given endovenously to rats and, after six hours, gluconeogenesis from alanine and several related parameters were evaluated in the isolated haemoglobin-free perfused rat liver. Livers from rats injected with TNFalpha and IL1beta, either alone or in combination, presented diminished gluconeogenesis. The degrees of inhibition caused by TNFalpha+IL1beta, TNFalpha and IL1beta were, respectively, 48.5, 38.8 and 30.4%. TNFalpha also diminished oxygen uptake. No action on urea and ammonia production was found. Possibly, both TNFalpha and IL1beta contribute to the decreased rates of hepatic gluconeogenesis that were found in rats with arthritis, sepsis and some kinds of cancer, but not to the decreased rates of ureagenesis.

Metabolic effects of p‐coumaric acid in the perfused rat liver

The p-coumaric acid, a phenolic acid, occurs in several plant species and, consequently, in many foods and beverages of vegetable origin. Its antioxidant activity is well documented, but there is also a single report about an inhibitory action on the monocarboxylate carrier, which operates in the plasma and mitochondrial membranes. The latter observation suggests that p-coumaric acid could be able to inhibit gluconeogenesis and related parameters. The present investigation was planned to test this hypothesis in the isolated and hemoglobin-free perfused rat liver. Transformation of lactate and alanine into glucose (gluconeogenesis) in the liver was inhibited by p-coumaric acid (IC50 values of 92.5 and 75.6 microM, respectively). Transformation of fructose into glucose was inhibited to a considerably lower degree (maximally 28%). The oxygen uptake increase accompanying gluconeogenesis from lactate was also inhibited. Pyruvate carboxylation in isolated intact mitochondria was inhibited (IC50 = 160.1 microM); no such effect was observed in freeze-thawing disrupted mitochondria. Glucose 6-phosphatase and fructose 1,6-bisphosphatase were not inhibited. In isolated intact mitochondria, p-coumaric acid inhibited respiration dependent on pyruvate oxidation but was ineffective on respiration driven by succinate and beta-hydroxybutyrate. It can be concluded that inhibition of pyruvate transport into the mitochondria is the most prominent primary effect of p-coumaric acid and also the main cause for gluconeogenesis inhibition. The existence of additional actions of p-coumaric acid, such as enzyme inhibitions and interference with regulatory mechanisms, cannot be excluded.

The hemodynamic effects of diltiazem in the isolated perfused rat liver are Ca(2+)-dependent.

Diltiazem reduces systemic blood pressure by decreasing the vascular smooth muscle tone. In the liver however, diltiazem seems to cause vasoconstriction, as evidenced by increases in portal pressure. The questions raised by this observation are concerned with a) the site of action of diltiazem (large vessels or sinusoids), b) the formation of permeability barriers and c) the role of Ca2+. The experiments in the present study should provide an answer to these questions. The experimental system was the hemoglobin-free perfused rat liver. The multiple-indicator dilution technique was employed with simultaneous injection of [14C]sucrose and [3H]water. Mean transit times and distribution spaces were calculated from the normalized outflow profiles. Calcium alone did not affect the hemodynamics of the liver. Diltiazem, however, changed several hemodynamic parameters when Ca2+ was present, but it was inactive in the absence of this cation. The hemodynamic effects of 500 microM diltiazem were: a) diminution of the transit time through the large vessels (t(o)) and, consequently, of the accessible vascular space (66.9%); b) diminution of the mean transit time of [14C]sucrose (tsuc) and, consequently, of the accessible sinusoidal space (28.1%); c) diminution of the mean transit time of tritiated water (twater) and, consequently, of the accessible cellular space (68.9%); d) diminution of the cellular to extracellular space ratio (theta) from 1.42 +/- 0.05 to 0.46 +/- 0.11. The linear superposition of the tritiated water and labeled sucrose curves, predicted by Goresky's model, could be optimized even when the curves were obtained with diltiazem + Ca2+, indicating that the distribution of both tracers was still flow-limited. The hemodynamic effects of diltiazem seem to be restricted to a vasoconstriction of the great vessels, an action which was strictly dependent on Ca2+. At the concentration of 500 microM, the effects of diltiazem were pronounced to the point of excluding completely about 2/3 of the liver parenchyma from the microcirculation, as indicated by the observed reduction in the accessible cell space. The sinusoids that were still supplied with perfusion fluid suffered considerable distension (2.19 fold) because the whole perfusate flow was deviated into the remaining 1/3 microcirculatory units. Diltiazem did not seem to induce the formation of intrahepatic shunts or diffusion barriers in the liver.

The role of Ca2+ and hemodynamics in the action of diltiazem on hepatic energy metabolism.

Diltiazem causes vasoconstriction in the liver when present at high concentrations, an action that is strictly Ca2+-dependent. Diltiazem is also active on energy metabolism. This toxic action could be partly a consequence of hemodynamic effects. In the absence of Ca2+, the hemodynamic effects are no longer present and, consequently, Ca2+-free experiments are useful for distinguishing between hemodynamics-dependent and hemodynamics-independent effects. The experimental system used was the hemoglobin-free perfused rat liver from fed and fasted rats. Diltiazem was infused at various concentrations in the presence and absence of Ca2+. Several metabolic parameters were measured: lactate and pyruvate production (glycolysis), glycogenolysis, oxygen uptake, gluconeogenesis, and the cellular levels of lactate, pyruvate, glucose, AMP, ADP, and ATP. The effects of diltiazem can be divided into three groups: (1) Effects that are strictly dependent on the Ca2+-mediated hemodynamic action. This group comprises inhibition of oxygen uptake at all concentrations (50-500 micromol/L) inhibition of lactate, pyruvate, and glucose release at high concentrations; the decrease in cellular ATP; the increase in cellular AMP; and the cellular accumulation of glucose and lactate. (2) Effects that are independent of the hemodynamic action. The most relevant effect of this type is inhibition of gluconeogenesis. (3) Effects that are influenced by Ca2+ but are independent of the hemodynamic effects. This is the typical case of lactate and glucose release from endogenous glycogen, whose stimulation by low diltiazem concentrations is more pronounced in the presence of Ca2+ than in its absence.

Some Characteristics of the Fluorescence Lifetime of Reduced Pyridine Nucleotides in Isolated Mitochondria, Isolated Hepatocytes, and Perfused Rat Liver In Situ

By extensively examining the experimental conditions for time-resolved spectrophotometry of non-transparent light scattering systems, we demonstrated the feasibility of quantitative analysis of both the fluorescence lifetime and intensity of reduced pyridine nucleotides in living tissues, suspensions of isolated liver mitochondria, and hepatocytes, as well as hemoglobin-free perfused rat liver being used systematically for measurements. The fluorescence decay was analyzed by the maximum likelihood method with a 4-component decay model. The lifetime of NADH observed in mitochondria (mean: 2.8 +/- 0.2 ns) was much longer than that of the free form in an aqueous solution (mean: 0.43 +/- 0.01 ns), and it was characterized as a protein-bound form. The lifetime was not affected by either aerobic or anaerobic conditions nor by the energy state, though the intensity changed markedly. The decay curves of isolated hepatocytes under normal aerobic conditions were the same as those of isolated mitochondria, though cytosolic NADH and NADPH were superimposed. Under the conditions of "unphysiological" acidosis, the mean lifetime became about 1.5 times longer than that under normal conditions. With perfused liver, the relative contributions of cytosolic NADH and NADPH were determined by infusing lactate and tert-butylhydroperoxide. Cytosolic NADH did not contribute to the overall fluorescence of pyridine nucleotides. In contrast, about 70% of the total fluorescence intensity was due to cytosolic NADPH, but its decay parameters were essentially the same as those of mitochondrial NADH. No free form of either NADH or NADPH was detected in the cytosolic and mitochondrial spaces. We concluded that the changes in fluorescence intensity observed under the various conditions can be simply explained by a change in the amount of reduced pyridine nucleotides in tissues, rather than by changes in the microscopic environment. The wide applicability of time-resolved fluorescence photometry to in vivo studies is well documented.

Microspectroscopic measurement of the optical properties of rat liver in the visible region

Microspectroscopy is used to investigate optical properties of haemoglobin-free perfused rat liver. Visible spectra of 20 microns diameter spot size were measured in transmission and/or reflection modes as a function of the thickness (< 1200 microns) of the liver-edge. Optical density (OD) in transmission mode increased with the increasing liver thickness, whereas in reflection mode OD decreased but became almost constant above a certain thickness (c.600 microns) of the liver. The Kubelka-Munk (KM) two-flux model, with a minor modification, was applied successfully to the analysis of the changes in OD as a function of the thickness. This approach estimates the KM absorption coefficient (EKM), KM scattering coefficient (SKM) and effective penetration depth (delta eff) of the liver. The optical properties were similar to reported values, obtained with different methods.

Zonation of gluconeogenesis from lactate and pyruvate in the rat liver studied by means of anterograde and retrograde bivascular perfusion

Gluconeogenesis from lactate and pyruvate and associated parameters were investigated in the bivascularly and hemoglobin-free perfused rat liver. The substrates were infused either via the portal vien (anterograd perfusion mode), via the hepatice vein (retrograde mode) or via the hepatic artery (anterograde and retrograde modes). The rates of lactate and pyruvate infusion were 10.3 and 3.5 ωmol min −1 g −1, respectively. The metabolic rates measured when the substrates were infused into the hepatic artery were referred to the cellular spaces accessible in each perfusion mode. The following results were obtained when the substrates were infused into the hepatic artery: (1) gluconeogenesis from lactate was equal to 2.08 ± 0.2 ωmol min −1 ml −1 in the retrograde mode and 1.33 ± 0.08 ωmol min − ml − in the anterograde mode ( P = 0.019); (2) gluconeogenesis from pyruvate was equal to 0.66 ± 0.11 ωmol min −1 ml −1 in the retrograde mode and 0.7 ± 0.11 ωmol min −1 ml −1 in the anterograde mode ( P = 0.75); (3) oxygen uptake increase with lactate was 1.75 ± 0.14 ωmol min −1 ml −1 in the retrograde mode and 1.05 ± 0.07 ωmol min −1 ml −1 in the anterograde mode ( P = 0.002); (4) oxygen uptake increase with pyruvate was equal to 0.59 ωmol min −1 ml −1 in the retrograde mode and 0.53 ± 0.05 ωmol min −1 ml −1 in the anterograde mode ( P = 0.75); ()5) pyruvate production from lactate was 0.28 ± 0.06 ωmol min −1 ml −1 in the retrograde mode and 0.39 ± 0.05 ωmol min −1 ml −1 in the anterograde monde ( P = 0.28); (6) lactate production from pyruvate was equal to 0.52 ± 0.05 ωmol min −1 ml −1 in the retrograde mode and 0.99 ± 0.08 ωmol min −1 ml −1 in the anterograde mode ( P < 0.001). Since only periportal cells are supplied with substrates when they are infused via the hepatic artery in retrograde perfusion, these results allow the conclusion that gluconeogenesis from lactate predominates in periportal hepatocytes. When pyruvate in the sole substrate, however, gluconeogenesis in periportal and perivenous cells presents no differences.

The metabolism of fructose in the bivascularly perfused rat liver

The metabolism of fructose was investigated in the bivascularly and hemoglobin-free perfused rat liver. Anterograde and retrograde perfusions were performed. In anterograde perfusion, fructose was infused at identical rates (19 μmol min −1 g −1) via the portal vein (all liver cells) or the hepatic artery (predominantly perivenous cells); in retrograde perfusion fructose was infused via the hepatic vein (all liver cells) or the hepatic artery (only periportal cells). The cellular water spaces accessible via the hepatic artery were measured by means of the multiple-indicator dilution technique. The following results were obtained, (i) Fructose was metabolized to glucose, lactate and pyruvate even when this substrate was infused via the hepatic artery in retrograde perfusion: oxygen consumption was also increased. (ii) When referred to the water spaces accessible to fructose via the hepatic artery in each perfusion mode, the rate of glycolysis was 0.99 ± 0.14 μmol min −1 ml −1 in the retrograde mode; and, 2.05 ± 0.19 μmol min −1 ml −1 in the retrograde mode ( P = 0.002). (iii) The extra oxygen uptake due to fructose infusion via the hepatic artery was 1.09 ± 0.16 μmol min −1 ml −1 in the retrograde mode; and, 0.51 ± 0.18 μmol min −1 ml −1 in the anterograde mode ( P = 0.005). (iv) Glucose production from fructose via the hepatic artery was 2.18 ± 0.18 μmol min −1 ml −1 in the retrograde mode; and, 1.83 ± 0.16 μmol min −1 ml −1 in the anterograde mode ( P = 0.18). (v) Glucose production and extra oxygen uptake due to fructose infusion did not correlate by a single factor in all perfusion modes. It was concluded that: (a) rates of glycolysis are lower in the periportal area, confirming previous views; (b) extra oxygen uptake due to fructose infusion is higher in the periportal area; (c) a predominance of glucose production in the periportal area could not be demonstrated; and (d) extra oxygen uptake due to fructose infusion is not a precise indicator for glucose synthesis.

Histological Detection of Lipid Peroxidation Following Infusion of Tert-Butyl Hydroperoxide and ADP-Iron Complex in Perfused Rat Livers.

Lipid peroxidation was assessed histologically and biochemically in hemoglobin-free perfused rat livers using two different types of stimulators. The Schiff reaction of fuchsin with cellular aldehydes was used as a histological index for lipid peroxidation. t-Butyl hydroperoxide (BHP, 0.8 mM) infusion caused a rapid and sustained release of thiobarbituric acid reactive substances (TBARS) into the effluent perfusate for up to 60 min, which was accompanied by lactate dehydrogenase (LDH) leakage after 30 min. The Schiff positive foci were initially restricted to periportal zones and spread with time to whole areas, accompanied by periportal necrosis. Coinfusion of diphenyl-p-phenylenediamine suppressed the TBARS release, with negative fuchsin staining, but the LDH leakage was unaffected. Under retrograde perfusion, BHP produced pericentral staining and necrosis. With 2.5 mM ADP-100 microM Fe3+, little TBARS was released up to 60 min, even though the hepatic TBARS levels increased considerably by this time. By 90 min, marked TBARS release occurred, but LDH leakage remained low. Irrespective of the direction of perfusion, pericentral hepatocytes became Schiff positive after 30 min. The fuchsin staining method may be useful for detecting peroxidized zones of the liver lobules.

Oxidative stress as a precursor to the irreversible hepatocellular injury caused by hyperthermia

Heat-induced hepatotoxicity accompanying hyperthermic liver perfusion was studied in the isolated, haemoglobin-free perfused rat liver. Trypan blue uptake, a sensitive indicator of cell death, was used to examine the relationship between the efflux of oxidized glutathione (oxidative stress), the appearance of cytosolic enzymes in the perfusate and cell death. Livers were perfused at 37, 42, 42.5 and 43 degrees C. The efflux of total glutathione (GSH) and oxidized glutathione (GSSG) increased with time and temperature. Differences between temperature groups were significant for both parameters for 37 versus 42, 42.5 and 43 degrees C (p less than 0.05). Temperature-related differences in GSH levels appeared at 15 min for 37 versus 42 degrees C and in GSSG levels at 30 min for 37 versus 42 and 42.5 degrees C. Biliary excretion of total GSH increased from 72 nmol at 37 degrees C to 144 nmol at 42 degrees C, 160 nmol at 42.5 degrees C and 124 nmol at 43 degrees C, which was significant for 37 versus 42 and 42.5 degrees C (p less than 0.05). The release of allantoin into the perfusate, a measure of purine catabolism and flux through xanthine oxidase, was increased at 42, 42.5 and 43 degrees C compared to 37 degrees C (p less than 0.05). Liver injury was assessed by measuring the release of asportate aminotransferase (AST) and lactate dehydrogenase (LDH) and uptake of trypan blue after perfusion at each temperature. There was a pronounced release of LDH and AST into the perfusate after 60 min of perfusion at 42, 42.5 and 43 degrees C, the levels of which were significantly different from the 37 degrees C mean level. There was no uptake of trypan blue after 60 min perfusion at 37 degrees C. Perfusion at 42, 42.5 and 43 degrees C resulted in the uptake of trypan blue in the pericentral areas, but the dye uptake was significant (p less than 0.05) compared to 37 degrees C at 42.5 and 43 degrees C only. These data show that heat-induced pericentral cell death is minimal after 60 min at 42-43 degrees C, and that the biochemical process which occurred during this period suggest 'oxidative stress' as a causative factor in hyperthermic hepatotoxicity. In addition, this liver toxicity is probably related to xanthine oxidase activity or the depletion of GSH as the initiating event which leads to lipid peroxidation and cellular damage.

Histological Detection of Lipid Peroxidation Following Infusion of Tert-Butyl Hydroperoxide and ADP-Iron Complex in Perfused Rat Livers

Lipid peroxidation was assessed histologically and biochemically in hemoglobin-free perfused rat livers using two different types of stimulators. The Schiff reaction of fuchsin with cellular aldehydes was used as a histological index for lipid peroxidation. t-Butyl hydroperoxide (BHP, 0.8 mM) infusion caused a rapid and sustained release of thiobarbituric acid reactive substances (TBARS) into the effluent perfusate for up to 60 min. which was accompanied by lactate dehydrogenase (LDH) leakage after 30 min. The Schiff positive foci were initially restricted to periportal zones and spread with time to whole areas, accompanied by periportal necrosis. Coinfusion of diphenyl-p-phenylenediamine suppressed the TBARS release, with negative fuchsin staining, but the LDH leakage was unaffected. Under retrograde perfusion. BHP produced pericentral staining and necrosis. With 2.5 mM ADP-100 μM Fe3+, little TBARS was released up to 60 min, even though the hepatic TBARS levels increased considerably by this time. By 90 min, marked TBARS release occurred, but LDH leakage remained low. Irrespective of the direction of perfusion, pericentral hepatocytes became Schiff positive after 30 min. The fuchsin staining method may be useful for detecting peroxidized zones of the liver lobules.

Enhancement of acute ethanol hepatotoxicity under conditions of low oxygen supply and ischemia/reperfusion: The role of oxygen radicals

Using isolated hemoglobin-free perfused rat livers we studied the effect of low oxygen supply on ethanol hepatotoxicity in two models. In the first model resembling low blood supply, perfusion rate was lowered from 60 to 1O ml/min after a 30 min-equilibration phase and kept low for 60 min. As a consequence, oxygen consumption fell from 1.76 + 0.15 μmol/min/g to 0.51 ± 0.02 μmol/min/g. In the second model, total ischemia was accomplished by interruption of the perfusion for 30 min and was followed by reperfusion at a perfusion rate of 60 ml/min for a further 30 min. In this model, oxygen consumption returned immediately to normal values upon reperfusion. In both models, low oxygen supply had no toxic effects of its own on livers from fed rats. While ethanol (3 g/1) given under normoxic conditions led to a moderate hepatotoxicity, its application in both models of partial as well as total ischemia and reperfusion resulted in a marked liver damage as evidenced by a strong release of sorbitol dehydrogenase, glutamate-pyruvate-transaminase, lactate dehydrogenase and glutathione, as well as by an increase in hepatic calcium content. Inhibition of ethanol metabolism by 4-methylpyrazol prevented liver damage in both models indicating that metabolism of ethanol is a prerequisite for its toxicity to occur. Also, hepatotoxicity was inhibited partially by catalase and Superoxide dismutase and nearly totally by deferrioxamine and allopurinol. Thus, reactive oxygen species which are produced during ethanol metabolism as well as under conditions of low oxygen supply are mediators of hepatic damage in both models employed.

Ubiquitous formation of catalase compound II in hemoglobin-free perfused rat liver and detection of novel spectral species

Spectral examinations of hemoglobin-free perfused rat livers with a high-sensitivity reflectance spectrophotometer have revealed an accumulation of catalase Compound II to an amount comparable to that of Compound I under the aerobic steady state. This finding is in contrast to a recent proposal that NADPH associated with catalase both prevents and reverses the accumulation of Compound II ( Kirkman, H. N., Galiano, S., and Gaetani, G. F. (1987) J. Biol. Chem. 262, 660–666 ). Furthermore, spectral species with a broad peak extending from 550 to 600 nm were observed in a time range between the methanol-induced decays of Compound I and Compound II. When rats were treated with 3-amino-1,2,4-triazole, catalase Compound I was not detected but spectral species with peaks at 570, 556 and 530 nm were observed. These novel spectral profiles suggest contributions from “peroxy” and “ferryl” forms of cytochrome oxidase.

Enhancement of hypoxic liver damage by ethanol: Involvement of xanthine oxidase and the role of glycolysis

Using isolated hemoglobin-free perfused rat livers we investigated the hepatotoxic effects of hypoxia, ethanol or the combination of both. Hypoxia only (90 min) led to a weak toxicity as evidenced by the efflux of the enzymes glutamate-pyruvate-transaminase (GPT) and sorbitol dehydrogenase (SDH). This toxic effect was slightly higher in livers treated with ethanol (3 g/l) under normoxic conditions. Ethanol added under hypoxic conditions, however, showed a strong hepatotoxic effect. Under hypoxic conditions, lactate + pyruvate production was increased fivefold over control, indicating that glycolysis was more effectively undergone as main source of energy. Addition of ethanol suppressed this effect, indicating that ethanol inhibited glycolysis. These results indicate that ethanol potentiates hypoxic liver damage by inhibiting the main metabolic pathway yielding ATP under low oxygen tension resulting in a severe energy deficit. Allopurinol (100 mg/l) inhibited the toxic effects seen with ethanol + hypoxia. Also, the inhibitory action of ethanol on glycolysis was antagonized. Our results are consistent with the following model: hypoxia converts NAD-dependent xanthine dehydrogenase (XD) into the oxygen-dependent xanthine oxidase (XO). Due to hypoxia and ethanol, purine metabolites and acetaldehyde accumulate and are metabolized via XO. This process leads to the production of oxygen radicals which most probably mediate both the inhibition of glycolysis and the direct toxic effects towards liver cells.

Influence of extracellular calcium on allyl alcohol-induced hepatotoxicity.

The role of calcium in allyl alcohol-induced hepatotoxicity was investigated in the isolated haemoglobin-free perfused rat liver. At a Ca++ concentration of 2.5 mmol/l in the perfusate, allyl alcohol (initial concentration 1.17 mmol/l) produced an enhanced release of GPT and SDH from the liver, an increase in the lactate/pyruvate ratio of the perfusate, a decrease in hepatic oxygen consumption and an increase of both hepatic calcium and malondialdehyde content. In the absence of Ca++ in the perfusate, no hepatic calcium accumulation occurred with allyl alcohol, but all other signs of hepatic damage were as severe as with 2.5 mmol/l Ca++. On the other hand, high extracellular Ca++ (5 mmol/l) alone led to a threefold increase of liver calcium but produced only marginal hepatotoxicity and only slightly enhanced the hepatotoxic effects of allyl alcohol. The concentrations of allyl alcohol in the perfusate were not altered at different Ca++ concentrations. In conclusion, the primary allyl alcohol-induced hepatotoxic injury does not appear to depend upon an influx of extracellular calcium.

Direct effect of carbon monoxide on hexobarbital metabolism in the isolated perfused liver in the absence of hemoglobin.

The interaction of carbon monoxide (CO) with cytochrome P-450 associated with hexobarbital metabolism was observed in hemoglobin-free perfused rat liver by using a scanning reflectance spectrophotometer. The evidence obtained showed that CO bound to the substrate complexed cytochrome P-450 and, at a CO/O2 ratio of over 0.1 in the perfusate, inhibited the hexobarbital metabolism estimated from the hexobarbital uptake, and oxygen consumption. Although the oxygen supply to the liver cell was one of the major limiting factors during CO hypoxia, CO binding to cytochrome P-450 significantly enhanced the suppression of hexobarbital oxidation caused by hypoxic hypoxia.

Chemically induced antioxidant-sensitive respiration. Relation to glutathione content and lipid peroxidation in the perfused rat liver.

The interrelations between the hepatic chemically induced antioxidant-sensitive respiration and the contents of malondialdehyde (MDA) and of reduced glutathione (GSH), were studied in the isolated hemoglobin-free perfused rat liver. Antioxidant-sensitive respiration was induced by the infusion of agents such as ethanol, iron, xanthine or t-butyl hydroperoxide, or by phenylhydrazine pretreatment in vivo. The development of this respiratory component occurred concomitantly with high levels of MDA in the perfused livers, while those of GSH were diminished.

Bile secretion in hemoglobin-free perfused rat liver.

Hemoglobin-free perfused rat liver was demonstrated to be a suitable experimental model in studying bile secretion. Bile flow slowly decreased to more than 3 h of perfusion. Despite differences in metabolic states, the bile flow was the same in the recirculating as in the nonrecirculating mode of perfusion. Sulfobromophthalein stimulated bile flow at high rates of infusion. In bile, the ratio conjugated to unconjugated sulfobromophthalein also increased with sulfobromophthalein infusion rate. The access of [14C]insulin, [14C] sucrose, and inorganic [32P] phosphate from perfusate into bile was restricted. Bile flow, secretion of taurocholate and sulfobromophthalein, and bile pressure are compared with values from anesthetized animals and from isolated livers perfused with medium containing erythrocytes.

Measuring rates of O2 uptake in periportal and pericentral regions of liver lobule: stop-flow experiments with perfused liver.

Rates of oxygen uptake in periportal and pericentral regions of the hemoglobin-free perfused rat liver were measured successfully for the first time with a miniature oxygen electrode (tip diameter, 50-60 microns) by stopping the flow of perfusate and measuring the rate of decrease of oxygen concentration on the surface of the liver. Rates of oxygen uptake in periportal and pericentral areas were 131 +/- 9 (mean +/- SD) and 56 +/- 14 mumol X g-1 X h-1 in livers from fed, phenobarbital-treated rats, respectively. In livers from fasted rats, rates of O2 uptake of 141 +/- 12 and 89 +/- 11 mumol X g-1 X h-1 were observed in periportal and pericentral regions, respectively. Similar data were obtained in livers from normal rats. Thus, periportal hepatocytes have higher rates of oxygen uptake than pericentral cells in both the fed and fasted states. Rates of oxygen uptake were not affected in periportal regions by fasting. In contrast, rates of oxygen uptake by hepatocytes in pericentral areas were significantly (P less than 0.001) greater in livers from fasted than from fed rats. This significant increase in oxygen uptake in pericentral hepatocytes as a result of fasting is consistent with the hypothesis that glycolysis occurs predominantly in pericentral regions of the liver lobule.

Periportal and pericentral pyridine nucleotide fluorescence from the surface of the perfused liver: evaluation of the hypothesis that chronic treatment with ethanol produces pericentral hypoxia.

Pyridine nucleotide fluorescence made from the surface of the hemoglobin-free perfused rat liver was measured continuously by using a "micro-light guide" placed on selected periportal and pericentral regions of the liver lobule. From the portal oxygen tension at which pyridine nucleotide reduction first occurred in pericentral regions, the oxygen gradient across the liver lobule was estimated in livers from rats treated chronically with ethanol or sucrose. Chronic treatment with ethanol increased the average lobular oxygen gradient from 275 to 400 torr (1 torr = 133 Pa), primarily due to the increase in the oxygen gradient in pericentral regions. Ethanol treatment also increased hepatic oxygen uptake significantly, from 110 to 144 (mumol/g)/hr. Treatment with the antithyroid drug 6-propyl-2-thiouracil reversed the effect of ethanol on O2 uptake and on the lobular oxygen gradient. The oxygen gradients measured with the micro-light guide were confirmed by direct measurement of tissue oxygen tensions in periportal and pericentral areas by using an oxygen electrode. These data are consistent with the hypothesis that chronic treatment with ethanol causes the pericentral region of the liver lobule to become susceptible to hypoxic cellular injury. This may be responsible, at least in part, for the localized hepatotoxic effects of ethanol.