Hepatic ATP Levels Research Articles

ISCHEMIC PRECONDITIONING PROTECTS POST-ISCHEMIC OXIDATIVE DAMAGE TO MITOCHONDRIA IN RAT LIVER

This study examined the effect of ischemic preconditioning (IPC) in protecting against a hepatic ischemia/reperfusion (I/R) injury, with particular focus on mitochondrial damage. Rat liver was preconditioned by 10 min of ischemia and 10 min of reperfusion. Immediately after IPC, liver was subjected to 90 min of sustained ischemia followed by 5 h of reperfusion. The hepatic I/R increased serum aminotransferase activity and mitochondrial lipid peroxidation 5 h after reperfusion. IPC attenuated these increases. Whereas the mitochondrial glutathione content and glutamate dehydrogenase activities were lower in the I/R group, these decreases were attenuated by IPC. During IPC, the tissue peroxide levels increased after 10 min of ischemia and were normalized after 10 min of reperfusion. In association with the IPC-derived transient increase in the peroxide levels, the significant production of peroxides observed at 10 min of reperfusion after 90 min of ischemia was attenuated. Furthermore, whereas the mitochondria isolated from rat liver after 5 h of reperfusion were rapidly swollen, the swelling rate was attenuated in the mitochondria from rat liver subjected to IPC before the sustained ischemia. The hepatic ATP and adenosine levels were 38% and 46% lower during the reperfusion, respectively. These decreases were attenuated by IPC. Thus, these results suggest that IPC protects the mitochondria against the deleterious effects of I/R, and this protection is associated with the reduced oxidative stress.

The Effect of Amrinone on Liver Regeneration in Experimental Hepatic Resection Model 1

The phosphodiesterase inhibitors (PDEIs) have been proposed to improve hepatic reperfusion injury and hepatosplanchnic circulation, but the effects of these agents on liver regeneration have not been investigated thoroughly. The aim of this study was to investigate the effect of amrinone, a PDEI, on liver regeneration in rats. Sixty rats were divided into two groups, control and amrinone. Each group was then divided into three groups (n=10). An infusion of amrinone to the study group and of 0.9% NaCl to the control group was performed. Seventy percent liver resection was performed to the rats during the first hour of infusion. The infusion was maintained for 17 h after resection. A total of 18 h infusion was performed. Rats were allowed to survive for 24, 48, and 72 h, and then they were sacrificed. Biochemical, morphological, hematological, and histopathologic measurements and assessments were performed. There were statistically significant differences between the amrinone and control groups in alkaline phosphatase and relative liver weights at 24, 48, and 72 h (P<0.05). There also were statistically significant differences between the groups in AST, bilirubin, and albumine levels at 24 h, ALT and prothrombine time levels at 48 h, and aspartate aminotransferase and alanine aminotransferase levels at 72 h (P<0.05). Hepatic ATP levels, mitotic index, and proliferating cell nuclear antigen labeling index were significantly higher in amrinone group compared with control group at all three time intervals (P<0.05). Amrinone improves both morphological and functional liver regeneration after liver resection.

Inhibition of tumor necrosis factor-induced apoptosis in transgenic mouse liver expressing creatine kinase.

The mitochondrion acts as a pivotal decision center in many types of apoptotic responses. To clarify the effects of the enhanced mitochondrial function on tumor necrosis factoralpha (TNFalpha)-induced apoptosis, we studied hepatic injuries in transgenic mice whose livers express creatine kinase (CK). Mice fed a diet containing 10% creatine, came to accumulate phosphocreatine and to enhance hepatic ATP levels and mitochondrial oxidative phosphorylation activities. TNFalpha-mediated hepatic apoptosis in normally fed and Cr-feeding CK transgenic mice were assessed. TNFalpha and actinomycin D cause severe liver failure in normally fed transgenic mice, and in the wild-type mice. In contrast, no significant elevations in transaminase levels after injection were observed in Cr feeding transgenic mice. The disruption of the mitochondrial transmembrane potential at 2 h after TNFalpha injection, prior to ATP depletion, activation of caspase 3 like protease, and DNA fragmentation at 4-6 h after injection, were observed in normally fed transgenic mice. These were fully suppressed in Cr feeding transgenic mice. However, anti-Fas antibody-induced apoptosis was not inhibited in both groups. The results indicate that TNFalpha-induced apoptosis was inhibited in CK transgenic mice livers by maintaining mitochondrial function.

Gene expression profile analysis of regenerating liver after portal vein ligation in rats by a cDNA microarray system.

We assessed changes in gene expression of hypertrophied liver after portal vein ligation (PL) in a test group of rats compared to a control group, which had the same size liver but no PL. The portal veins of the left and median lobes in the test group were ligated in an initial operation. Four days after the PL, the liver volume of the posterior caudate lobe (5%) increased two-fold and comprised 10% of the liver. A 90% hepatectomy was then performed, leaving only the hypertrophied posterior caudate lobe, and leaving the normal anterior and posterior caudate lobes (10%) in the control (sham) group. A comparison of the expression profiles between two groups was performed using cDNA microarrays and the hepatic ATP level was measured. The survival rate for the PL group was significantly higher than for the sham group at 4 days after the hepatectomy (56.3% and 26.7%, P < 0.05). Gene expression of cyclin D1, proliferating cell nuclear antigen, cyclin A and B was upregulated, and the cyclin-dependent kinase inhibitor was downregulated. Increases were observed in: (i) pyruvate dehydrogenase, the tricarboxylic acid cycle cycle regulator, (ii) acyl-CoA dehydrogenase, the oxidation regulator, and (iii) cytochrome oxidases, the oxidative phosphorylation regulator. Hepatic ATP concentration after hepatectomy was better maintained in the PL group than in the sham group (0.48 +/- 0.01 micromol/ml vs. 0.33 +/- 0.01 micromol/ml, P < 0.05). The regenerating liver increased tolerance for extended hepatectomy compared to normal liver. It is believed that this is because the induced rapid regeneration of the remaining liver after hepatectomy increases ATP metabolism.

Effect of iron lactate overloading on adenine nucleotide levels and adenosine 3'-monophosphate forming enzyme in rat liver and spleen.

To elucidate the pathophysiological significance of adenosine 3'-monophosphate (3'-AMP) forming enzyme in rats, the effect of iron lactate overloading on the enzyme activities and adenine nucleotide levels in the liver and spleen was examined. Sprague-Dawley rats were fed a diet supplemented with 0%, 0.625% or 5.0% of iron lactate for 4 weeks. Iron deposition was found in periportal hepatocytes, Kupffer cells and macrophages of red pulp of the spleen. No significant changes in hematological parameters were detected. Although serum alkaline phosphatase and inorganic phosphorus levels elevated slightly in the 5.0% group, activities of alanine aminotransferase and aspartate aminotransferase, and levels of serum urea nitrogen and creatinine were not changed significantly. The ATP levels in the liver and spleen of iron fed groups were significantly decreased, but adenosine 5'-diphosphate (ADP) and adenosine 5'-monophosphate (AMP) levels were within control levels. On the other hand, the levels of ATP, ADP and AMP in the erythrocytes without mitochondria were not suppressed by the iron lactate overloading. Free activity of 3'-AMP forming enzyme, one of ribonucleases (RNase), was not changed in the liver of iron-overloaded rat, and total amount of 3'-AMP and adenosine formed after the treatment of the crude enzyme(s) with p-chloromercuribenzensulfonic acid, a SH blocker of RNase inhibitors, was decreased dose-dependently. On the contrary, free activity of 3'-AMP forming enzyme was enhanced dose-dependently in the spleen of iron-overloaded rat but the total activity was not changed. However, the free and total 3'-AMP forming enzyme activities in the liver and spleen of iron-overloaded rats became equal at the dosage of 5.0% of iron lactate. The results obtained suggested that iron loading might induce significant decrease in hepatic and splenic ATP levels via malfunction of their mitochondria and might lead dissociation of RNase-RNase inhibitor complex to activate 3'-AMP forming enzyme in both tissues.

Recovery of hepatocellular ATP and "pericentral apoptosis" after hemorrhage and resuscitation.

Progressive liver dysfunction contributes significantly to the development of multiple organ failure after trauma/hemorrhage. This study tested the relative impact of necrotic and apoptotic cell death in a graded model of hemorrhagic shock (mean arterial blood pressure=35+/-5 mmHg for 1, 2, or 3 h, followed by 2 h, 1 h, or no resuscitation, respectively) in rats. Prolonged periods of hemorrhagic hypotension (3 h) were paralleled by a profound decrease of hepatic ATP levels and occurrence of pericentral necrosis. Resuscitation after shorter periods of hemorrhagic hypotension resulted in restoration of tissue ATP whereas hepatocellular function as assessed by indocyanine green clearance remained depressed (49.9+/-1.6 mL/(min x kg) at baseline, 28.8+/-1.2 mL/(min x kg) after 2 h of resuscitation; P<0.05). Under these conditions, induction of caspase activity and DNA fragmentation were observed in pericentral hepatocytes that could be prevented by the radical scavenger tempol. Pretreatment with z-Val-Ala-Asp(O-methyl)-flouromethylketone prevented de novo expression of caspase-generated cytokeratin 18, DNA fragmentation, and depression of hepatocellular indocyanine green clearance. These data suggest that prolonged low flow/hypoxia induces ATP depletion and pericentral necrosis and restoration of oxygen supply and ATP levels after shorter periods of low flow ischemia propagate programmed cell death or "pericentral apoptosis."

Myoglobin gene expression attenuates hepatic ischemia reperfusion injury

Background.Cellular functions are maintained by a continuous supply of ATP, which is supplied efficiently by mitochondrial oxidative phosphorylation. Since myoglobin, found in cardiac myocytes and red skeletal muscle, but not in the liver, facilitates oxygen diffusion under low oxygen conditions and enhances oxidative phosphorylation, this study seeks to enhance hepatic ATP levels and attenuate ischemia-reperfusion injury in rodent livers by adenovirus-mediated myoglobin expression. Material and methods.After infecting Hep3B and rodent livers with adenovirus carrying CMV promoter sequences linked to the human myoglobin gene (AdCMVMyo), reverse transcriptase-PCR and immunodetection for myoglobin, and cellular and hepatic ATP levels were examined. The effect of myoglobin was evaluated in a hepatic ischemia-reperfusion model in the rat. Results.Myoglobin expression was confirmed in Hep3B and rat livers after AdCMVMyo infection. The ATP levels in Hep3B cells and C57BL/6 mice livers 72 h after AdCMVMyo transfection were significantly higher than control levels and those after adenovirus-mediated β-galactosidase transfection. Finally, expression of myoglobin attenuated ischemia-reperfusion injury in the rat liver. Conclusion.These results indicate that myoglobin gene transfer to the liver enhanced ATP levels both in vitro and in vivo and might be a novel strategy to reduce ischemia-reperfusion injury.

Relation of biliary bile acid output to hepatic adenosine triphosphate level and biliary indocyanine green excretion in humans.

We have previously demonstrated that there are two subgroups of patients with different types of biliary bile acid output after relief of obstructive jaundice by percutaneous transhepatic biliary drainage (PTBD). The reason for two groups is not clear but is possibly the difference in hepatic reserve function. The aim of this study was to examine the relation of biliary bile acid output to the hepatic ATP level and biliary excretion rate of indocyanine green (ICG) in humans. Patients whose bile could be collected through a PTBD tube participated in this study. The mean serum total bilirubin concentration was 12.7 mg/dl at the time of PTBD, decreasing to 1.1 mg/dl before surgery. These patients underwent curative resection for cancer of the bile duct, duodenal papilla, or pancreatic head after the relief of hyperbilirubinemia. Bile was collected at 1-hour intervals for 5 hours after intravenous administration of ICG (0.5 mg/kg) within a few days before surgery, and a small liver tissue sample was obtained immediately after laparotomy without using ischemic procedures. The concentrations of total bile acid and ICG in bile, the bile flow rate, and the bile acid output and ICG excretion rate in bile over 5 hours were determined. ATP concentrations in liver tissue were determined by high performance liquid chromatography. Results of hepatic ATP levels were correlated with the bile acid output and ICG excretion rate into bile. Both the biliary bile acid output (micromoles per 5 hours) and ICG excretion rate (percent of injected dose of ICG) over 5 hours were significantly correlated with the hepatic ATP level (p = 0.0190 and p = 0.0084, respectively). Neither the bile flow rate nor the serum liver function tests were related to the hepatic ATP level. Significant correlation was found between the bile acid output and the ICG excretion rate (p = 0.0127). Biliary bile acid output reflects the hepatic ATP level. Determination of the biliary bile acid output and ICG excretion may provide useful parameters for evaluating the hepatic energy status, which is essential for organ viability.

S-adenosylmethionine protects post-ischemic mitochondrial injury in rat liver

Background/Aims: S-adenosylmethionine (SAM) is a thiol-containing compound with known therapeutic affects on cholestasis and hepatotoxicity. The aim of this study was to investigate the effect of SAM on the prevention of mitochondrial injury induced by hepatic ischemia and reperfusion.Methods/Results: Rats were subjected to 60 min of hepatic ischemia and 1 and 5 h of reperfusion. 2 h prior to ischemia, the animals received either vehicle or SAM intraperitoneally. In the vehicle-treated ischemic animals, serum aspartate aminotransferase levels increased at 1 h and again at 5 h of reperfusion and were reduced by SAM pre-treatment. Similarly, mitochondrial lipid peroxidation was elevated in the vehicle-treated group, but this elevation was attenuated by SAM. In contrast, mitochondrial glutamate dehydrogenase activity and reduced glutathione concentration both decreased in the vehicle-treated group, and this decrease was also inhibited by SAM. Hepatic ATP levels in the vehicle-treated rats were found to be 42% lower 5 h after reperfusion, however, treatment with SAM elevated these ATP levels. SAM treatment increased the concentration of adenosine but inhibited the accumulation of hypoxanthine in the ischemic liver.Conclusion: SAM protects against mitochondrial injury, which prevents mitochondrial oxidant stress and improves ischemia-induced hepatic energy metabolism.

Effect of S-adenosylmethionine on hepatic injury from sequential cold and warm ischemia.

We investigated whether S-adenosylmethionine (SAM) treatment improved ischemic injury using perfused rat liver after sequential periods of 24 h cold and 20 min re-warming ischemia. SAM (100 micromol/L) was added to University of Wisconsin (UW) solution and Ringers lactate solution. After cold and sequential warm ischemia, releases of lactate dehydrogenase (LDH) and purine nucleoside phosphorylase (PNP) markedly increased during reperfusion. The increase in PNP was significantly reduced by SAM treatment. While the concentration of reduced glutathione (GSH) in ischemic livers significantly decreased, the concentration of glutathione disulfide (GSSG) increased. This decrease in GSH and increase in GSSG were suppressed by SAM treatment. Lipid peroxidation was elevated in cold and warm ischemic and reperfused livers, but this elevation was also prevented by SAM treatment. Hepatic ATP levels were decreased in the ischemic and reperfused livers to 42% of the control levels. However, treatment with SAM resulted in significantly higher ATP levels and preserved the concentration of AMP in ischemic livers. Our findings suggest that SAM prevents oxidative stress and lipid peroxidation and helps preserve hepatic energy metabolism.

The beneficial effect of ATP–MgCl 2 on hepatic ischemia/reperfusion-induced mitochondrial dysfunction

The present study was undertaken to determine whether ATP–MgCl 2 administration in rats could protect hepatic mitochondrial function and improve energy metabolism during hepatic ischemia and subsequent reperfusion. Global hepatic ischemia was produced for 60 min followed by reperfusion. The rats then received 0.5 ml of saline or ATP–MgCl 2 intravenously. In saline-treated ischemic rats, serum alanine-aminotransferase levels peaked at 5 h. The aminotransferase level was significantly reduced in the ATP–MgCl 2 treatment group. The wet weight-to-dry weight ratio of the liver was significantly increased by ischemia/reperfusion. ATP–MgCl 2 treatment minimized the increase in this ratio. The ketone body ratio in blood, which reflects the mitochondrial free NAD +/NADH ratio, decreased after ischemia and at 1 h following reperfusion. This decrease was somewhat improved by ATP–MgCl 2 infusion. At 1 and 5 h after reperfusion, mitochondrial monoamine oxidase and glutamate dehydrogenase activities decreased. ATP–MgCl 2 infusion following ischemia restored the lost activities. Hepatic ATP levels in saline-treated rats were found to be 50% lower 5 h following reperfusion; however, treatment with ATP–MgCl 2 resulted in significantly higher ATP levels and energy charge. The accumulation of purine catabolites in ischemic tissues was reduced during reperfusion. ATP–MgCl 2 infusion resulted in accumulation of adenosine in reperfused liver. Mitochondrial lipid peroxidation was elevated in the saline-treated ischemic group, but this elevation was inhibited by ATP–MgCl 2 infusion. The present results lead us to conclude that the amelioration of liver function which occurs with ATP–MgCl 2 infusion following ischemia may be mediated through improvement in ischemia-induced mitochondrial energy metabolism.

The use of insulin and glucose during resuscitation from hemorrhagic shock increases hepatic ATP.

Hemorrhagic shock produces a marked decrease in hepatic ATP, adenylate energy charge, and total adenosine nucleotides. This is followed by slow recovery to normal levels after resuscitation. Nucleotide metabolites are increased following shock and resuscitation. Previous experimental work has shown that supraphysiologic doses of insulin have salutary effects in animals with hemorrhagic shock and in cardiac patients. It appears that insulin causes increased availability of glucose and energy-producing substrates. This study examined whether resuscitation with glucose and insulin after hemorrhagic shock would alter the changes previously seen to occur in hepatic ATP levels, adenylate energy charge, or nucleotide metabolites. Male Sprague-Dawley rats were bled to a mean arterial blood pressure of 40 mm Hg for 30 min. They were then resuscitated with the shed blood and one of three fluids: (1) lactated Ringer's, (2) lactated Ringer's with 10% glucose, (3) lactated Ringer's with 10% glucose + 6 units/kg regular insulin. Liver biopsies were obtained prior to shock (baseline), after 30 min of shock (shock), and 90 min after resuscitation (90 min). Tissue levels of ATP, ADP, AMP, adenosine, inosine, hypoxanthine, and xanthine were measured. Serum at 90 min was evaluated for potassium, glucose, and tumor necrosis factor alpha (TNF-alpha). The insulin-treated group had significantly increased hepatic ATP and energy charge following resuscitation compared with the other two groups. The insulin group also exhibited significant hypoglycemia. Total adenine nucleotides (ATP, ADP, and AMP) were significantly elevated 90 min postresuscitation in the insulin group. Mean blood pressures throughout the experiment were not significantly different among groups. TNF-alpha was highest in the insulin-treated group, but this was not significant. Resuscitation with insulin and dextrose significantly increased hepatic ATP and adenylate energy charge after hemorrhagic shock in rats. Total nucleotide pool levels were not different between groups, indicating that there was a shift of the equilibrium away from the metabolites toward ATP and ADP in the insulin-treated group. Insulin treatment had no significant effect on blood pressure or TNF-alpha. However, it caused significant hypoglycemia and hypokalemia.

The effects of dimethylsulfoxide on experimental hepatic ischemia.

The aim of the study is to investigate the effects of dimethylsulfoxide (DMSO), a non-enzymatic free oxygen radical detoxifier, on the alterations observed during hepatic ischemia. Twenty four albino rabbits were entered into the study. DMSO (500 mg/kg) was administered through inferior vena cava following dissection of the portal triad and immediately prior to clamping. The alterations on liver glycogen, blood glucose, ALT, AST, LDH, intracellular ATP, GSH-px, SOD, MDA within the erythrocyte and hepatic tissue MDA were investigated. In the control group, following ischemia, a reduction in blood glucose, hepatic glycogen and ATP levels within the cell and an increase in AST and LDH levels, MDA, GSH-px and SOD levels within the erythrocyte and hepatic tissue MDA level were observed (p < 0.01). In the study group, the blood glucose values increased at 30th minute following ischemia. The increase in LDH level was not statistically significant (p > 0.05). The increase in AST level was significantly lower when compared to the control group (p < 0.05). No effect was observed on ALT and ALP levels. DMSO lowered the reduction in hepatic glycogen level (p < 0.05), GSH-px (p < 0.01), MDA (p < 0.05) and ATP (p < 0.05) levels within the erythrocyte and hepatic tissue MDA level (p < 0.01) that were increased following ischemia. The increase in blood SOD level was not statistically significant (p > 0.05). DMSO can be administered in attempt to prolong the duration of ischemia or to reduce the adverse effects of ischemia on the hepatic tissue during the existing ischemic period.

Effects of Physical Exercise on Liver ATP Levels in Fasted and Phosphate-Injected Rats

The purpose of the present study was to investigate the effects of exercise (30 min, 23 m/min, 0% grade) on the hepatic levels of ATP in fasted adrenodemedullated rats, with an intraperitoneal injection of sodium phosphate (Na 2 PO 4, 0.91 mM) or saline (NaCl). Sodium phosphate was injected to determine if the postulated decrease in liver ATP during exercise may be changed by providing an excess of phosphate. At the end of exercise, a piece of liver was rapidly freeze clamped and used for the enzymatic determination of ATP levels. Liver ATP, in saline-injected rats, was significantly (P < 0.05) decreased by fasting, compared to fed rats (X ± SE: 3.21 ± 0.2 vs 2.86 ± 0.2 µmol/g). Exercise in fasted rats decreased even more the ATP response in liver (2.58 ± 0.14 µmol/g). Injection of Na 2 PO 4 did not significantly (P < 0.05) alter the pattern of ATP response following these 3 conditions (3.35 ± 0.14 vs 3.0 ± 0.12 vs 2.57 ± 0.1 µmol/g), ATP levels being significantly (P < 0.05) decreased by the fast and the exercise in the fasted state. Fasting and exercise resulted in a significant (P < 0.05) decrease in liver glycogen and plasma glucose concentrations and an increase in free fatty acid levels in both NaCl- and Na 2 PO 4 -injected groups. In both injection conditions, ß-hydroxybutyrate and peripheral insulin concentrations were respectively, increased and decreased (P < 0.05) by fasting, while norepinephrine and portal glucagon were decreased (P < 0.05) following exercise. The main effect of the injection of Na 2 PO 4 was a stimulation (P < 0.05) of peripheral glucagon response following exercise. It is concluded that exercise results in a decrease in liver ATP levels even in fasted rats and that this decrease is not corrected by Na 2 PO 4 administration. The decreased liver ATP levels might be involved in the metabolic adaptations to exercise.

The Beneficial Effect of Phosphocreatine Accumulation in the Creatine Kinase Transgenic Mouse Liver in Endotoxin-Induced Hepatic Cell Death

Purpose.The purpose of this study was to investigate the relationship between hepatic energy status and liver injury during sepsis, using transgenic mice which express creatine kinase in the liver catalyzing the phosphocreatine/creatine system.Methods.Creatine kinase transgenic mice were fed with normal rodent chow or chow containing 10% creatine for 5 days. Lipopolysaccharide (0.2 mg/kg) combined withd-galactosamine (600 mg/kg) was administered intraperitoneally.Results.Eighty percent of the creatine-fed transgenic mice had survived at 48 h post-d-galactosamine and lipopolysaccharide administration, compared with none of the normally fed transgenic mice. Hepatic phosphocreatine and ATP levels in the normally fed transgenic mice were significantly lower than those in the creatine-fed transgenic mice before and after lipopolysaccharide combined withd-galactosamine was administered. Massive hepatic hemorrhagic necrosis with apoptosis was seen in response tod-galactosamine and lipopolysaccharide in normally fed transgenic mice. These results are consistent with a significant increase in serum aminotransferase at 8 h. In contrast, there were faint necrotic changes in the liver with minimal cellular infiltration in creatine-fed transgenic mice.Conclusions.Maintenance of hepatic ATP levels protects from sepsis-induced liver injury and mortality.

INHIBITION OF THROMBOXANE A2 SYNTHETASE BY OKY-046 ATTENUATES ISCHEMIA AND REPERFUSION INJURY OF CANINE LIVERS

37 Protection against ischemia and reperfusion (I/R) injury of the liver is essential to achieve satisfactory outcome after liver transplantation. Although various factors have been proposed to explain the cause of hepatic I/R injury, derangement of eicosanoid metabolism, particularly suppressed production of prostagrandin I 2, and increase of thromboxane A 2 and leukotrienes, has a critical role. These changes have been shown to induce microcirculatory disturbance, neutrophils adherence to endothelial cells, and their activation. Using a 2-hour total hepatic vascular exclusion model in dogs, we tested our hypothesis that the regulation of eicosanoid metabolism by thromboxane A 2 synthetase inhibitor, OKY-046, would attenuate I/R injury of the liver. After skeletonization, the liver was made totally ischemic for two hours by cross-clamping the portal vein, the hepatic artery, the vena cava (above and below the liver). Veno-venous bypass was used to decompress splanchnic and inferior systemic venous congestion during procedure. Sixteen beagle dogs were divided into two groups: five animals were treated with OKY-046 (group OKY), and 11 received no treatment (group CT). In treated animals, OKY-046 was given intravenously at a rate of 50µg/kg/min for 60 minutes before ischemia and 3 hours after reperfusion. Animal survival, hepatic tissue blood flow(HTBF), portal vein pressure(PVP), liver enzymes and lactate levels, systemic hemodynamics, and hepatic tissue ATP level were determined. Histopathologically severity of liver tissue damage and the number of infiltrated polymorphonuclear cells were analyzed. TableTableCompared to the control, all treated animals by OKY-046 exhibited significantly better survivals, higher HTBF, earlier PVP restoration, and less hepatocyte's damage. No bleeding tendency related to the drug administration was noted. Ischemia and reperfusion injury of the liver was markedly attenuated by OKY-046, indicating it as a promising agent for the improvement of liver preservation.

Effects of platelet-activating factor antagonist E5880 on intrahepatic and systemic metabolic responses to transient hepatic inflow occlusion and reperfusion in the rabbit.

We investigated the effects of pretreatment with a potent platelet-activating factor (PAF) receptor antagonist (E5880) on the changes in hepatic and systemic metabolism induced by transient hepatic ischemia and reperfusion. Sixty-five rabbits were assigned to four groups that either did or did not undergo a period of hepatic ischemia and reperfusion with or without pretreatment. E5880 was administered intraportally 1 minute prior to inflow occlusion. Twenty minutes of warm ischemia was followed by 30 minutes of reperfusion. Blood gas analyses and measurements of levels of arterial pyruvate, lactate, and ketone bodies, arterial and portal ammonia and endotoxin, and intrahepatic adenine nucleotide, pyruvate, and lactate were performed. Results were analyzed by either ANOVA or chi-square analysis. Hepatic tissue ATP and energy charge levels were significantly increased and the AMP level was significantly decreased after 30 minutes of reperfusion in the pretreatment group compared to those without pretreatment. At the same time, parameters reflecting hepatic mitochondrial function, such as the arterial ketone body ratio and arterial ammonia level, improved, although they were not statistically significant. No difference was observed for parameters reflecting systemic changes, such as arterial blood gas values and pyruvate and lactate levels. PAF is thought to mediate metabolic changes after hepatic ischemia and reperfusion. PAF released in the liver may exert local effects, which appear to be attenuated by pretreatment with E5880. Systemic metabolic changes seen after hepatic ischemia and reperfusion may be mediated by factors other than PAF.

Involvement of Platelet Activating Factor in Microcirculatory Disturbances after Global Hepatic Ischemia

This study aimed at analyzing the involvement of platelet activating factor (PAF) in ischemia/reperfusion injury (I/R) of the liver. Male Wistar rats under pentobarbital anesthesia were subjected to 60 min of normothermic ischemia of the left and median liver lobes, followed by 30 min of reperfusion in vivo. Blood pressure and body temperature were controlled throughout the experiment. Preischemic injection of a specific PAF antagonist (BN52021,5 mg/kg body mass) resulted in significant reduction of postischemic enzyme loss into the serum from the vascular endothelium (purine nucleoside phosphorylase: 56.9 ± 11.4 vs 86.6 ± 20.4 U/I**) and the hepatic parenchyme (alanine aminotransferase: 176 ± 60 vs 519 ± 180 U/I***), accompanied by a significant increase of hepatic bile production (1.28 ± .32 vs 0.80 ± 0.16 μl/g/min*) and tissue levels of ATP (6.12 ± 1.73 vs 4.21 ± 1.30 μmol/g*). Laser Doppler flowmetry revealed a significant improvement by BN52021 of left lobular erythrocyte flux recovery from 27 ± 25 to 78 ± 19% of respective preischemic control values. The data give evidence for an implication of PAF in I/R damage to the vascular endothelium and in impaired parenchymal function of the liver, probably due to altered microvascular reperfusion. Treatment with PAF antagonists should improve results after liver surgery under ischemic conditions. (*; **; ***: P < 0.05; 0.01; 0.001)

Effects of Inhalation Anesthetics on Myocardial and Hepatic Energy Metabolism in Normotensive and Spontaneously Hypertensive Rats Subjected to Hemorrhage

Forty spontaneously hypertensive rats (SHRs) and forty normotensive Wistar-ST rats (NRs) were used to assess the influence of anesthetics on myocardial and hepatic energy metabolism after hemorrhage. They were divided into five pairs of groups: a control group (pentobarbital 6 mg.100 g BW-1 ip), and four others which received 1.2% halothane, 2.2% enflurane, 1.4% isoflurane, and 3.3% sevoflurane, respectively. Following a 10 min stabilization period, blood (2 ml.100 g BW-1) was gradually withdrawn over a 5 min period from a femoral artery. Thirty min after the induction of hemorrhage, the heart and liver were removed and myocardial and hepatic metabolites (ATP, lactate, pyruvate and glycogen) were measured by enzymatic methods. There were no significant differences in myocardial metabolites among either the anesthetic groups or between SHRs and NRs. However, hepatic ATP levels in all SHR groups were significantly lower than those in NR groups. Moreover, ATP levels in the inhalation anesthetic groups of SHRs were significantly higher than that in the control group of SHRs. All inhalation anesthetics, especially isoflurane, may reduce metabolic deterioration of the liver during hemorrhage when compared to barbiturate anesthesia.

Changes in Biliary Glutathione Level during Ischemia-Reperfusion of Rat Liver

Changes in hepatic and biliary glutathione levels were studied in rat liver treated with tert-butyl hydroperoxide (t-BuOOH) and subjected to ischemia-reperfusion. Immediately after t-BuOOH administration, the oxidized glutathione (GSSG) values and reduced glutathione (GSSG/GSH) ratio in the bile increased dose-dependently and then returned to control level within 10 min, whereas the hepatic ATP level and bile flow rate were not affected by t -BuOOH at doses of up to 1.0 mmol/kg. These data suggested that the liver remains viable on treatment with up to 1.0 mmole/kg t-BuOOH, and that hepatocytes can rapidly dismute t-BuOOH at up to this dose. The hepatic GSH and GSSG levels did not vary appreciably during isehemia for 10 or 30 min or during subsequent reperfusion, but the GSSG/GSH ratio increased after isehemia for 30 min. The rate of bile flow and the biliary level of GSH decreased after isehemia for 30 min in proportion to the decrease in the hepatic ATP level. However, the biliary GSSG concentration did not vary on reperfusion, although GSSG secretion into the bile is also related to the hepatic ATP level. As a result, the GSSG/GSH ratio in the bile increased during reperfusion after ischemia for 30 min. This increased ratio is thought to reflect oxidation of hepatic GSH by hydroperoxide produced during reperfusion. The GSSG/GSH ratio in the bile after 30 min ischemia corresponded to that observed after a small dose (0.07 mmole/kg body wt) of t-BuOOH, which hepatocytes could dismute rapidly without loss of their viability. Furthermore, the redox state of NADP (NADPH/[NADPH + NADP+]) remained constant during ischemia-reperfusion. These findings suggest that reactive oxygen was produced in the liver during the reperfusion after 30 min ischemia in amounts small enough to be scavenged by intrinsic protection mechanisms, including the NADP-glutathione system.