Hepatic Mitochondrial Redox State Research Articles

The PNPLA3 I148M variant increases ketogenesis and decreases hepatic de novo lipogenesis and mitochondrial function in humans

The PNPLA3 I148M variant is the major genetic risk factor for all stages of fatty liver disease, but the underlying pathophysiology remains unclear. We studied the effect of this variant on hepatic metabolism in homozygous carriers and non-carriers under multiple physiological conditions with state-of-the-art stable isotope techniques. After an overnight fast, carriers had higher plasma β-hydroxybutyrate concentrations and lower hepatic de novo lipogenesis (DNL) compared to non-carriers. After a mixed meal, fatty acids were channeled toward ketogenesis in carriers, which was associated with an increase in hepatic mitochondrial redox state. During a ketogenic diet, carriers manifested increased rates of intrahepatic lipolysis, increased plasma β-hydroxybutyrate concentrations, and decreased rates of hepatic mitochondrial citrate synthase flux. These studies demonstrate that homozygous PNPLA3 I148M carriers have hepatic mitochondrial dysfunction leading to reduced DNL and channeling of carbons to ketogenesis. These findings have implications for understanding why the PNPLA3 variant predisposes to progressive liver disease.

Distinct contributions of metabolic dysfunction and genetic risk factors in the pathogenesis of non-alcoholic fatty liver disease

Background & AimsThere is substantial inter-individual variability in the risk of non-alcoholic fatty liver disease (NAFLD). Part of which is explained by insulin resistance (IR) (‘MetComp’) and part by common modifiers of genetic risk (‘GenComp’). We examined how IR on the one hand and genetic risk on the other contribute to the pathogenesis of NAFLD.MethodsWe studied 846 individuals: 492 were obese patients with liver histology and 354 were individuals who underwent intrahepatic triglyceride measurement by proton magnetic resonance spectroscopy. A genetic risk score was calculated using the number of risk alleles in PNPLA3, TM6SF2, MBOAT7, HSD17B13 and MARC1. Substrate concentrations were assessed by serum NMR metabolomics. In subsets of participants, non-esterified fatty acids (NEFAs) and their flux were assessed by D5-glycerol and hyperinsulinemic-euglycemic clamp (n = 41), and hepatic de novo lipogenesis (DNL) was measured by D2O (n = 61).ResultsWe found that substrate surplus (increased concentrations of 28 serum metabolites including glucose, glycolytic intermediates, and amino acids; increased NEFAs and their flux; increased DNL) characterized the ‘MetComp’. In contrast, the ‘GenComp’ was not accompanied by any substrate excess but was characterized by an increased hepatic mitochondrial redox state, as determined by serum β-hydroxybutyrate/acetoacetate ratio, and inhibition of hepatic pathways dependent on tricarboxylic acid cycle activity, such as DNL. Serum β-hydroxybutyrate/acetoacetate ratio correlated strongly with all histological features of NAFLD. IR and hepatic mitochondrial redox state conferred additive increases in histological features of NAFLD.ConclusionsThese data show that the mechanisms underlying ‘Metabolic’ and ‘Genetic’ components of NAFLD are fundamentally different. These findings may have implications with respect to the diagnosis and treatment of NAFLD.Lay summaryThe pathogenesis of non-alcoholic fatty liver disease can be explained in part by a metabolic component, including obesity, and in part by a genetic component. Herein, we demonstrate that the mechanisms underlying these components are fundamentally different: the metabolic component is characterized by hepatic oversupply of substrates, such as sugars, lipids and amino acids. In contrast, the genetic component is characterized by impaired hepatic mitochondrial function, making the liver less able to metabolize these substrates.

Effect of a ketogenic diet on hepatic steatosis and hepatic mitochondrial metabolism in nonalcoholic fatty liver disease

Weight loss by ketogenic diet (KD) has gained popularity in management of nonalcoholic fatty liver disease (NAFLD). KD rapidly reverses NAFLD and insulin resistance despite increasing circulating nonesterified fatty acids (NEFA), the main substrate for synthesis of intrahepatic triglycerides (IHTG). To explore the underlying mechanism, we quantified hepatic mitochondrial fluxes and their regulators in humans by using positional isotopomer NMR tracer analysis. Ten overweight/obese subjects received stable isotope infusions of: [D7]glucose, [13C4]β-hydroxybutyrate and [3-13C]lactate before and after a 6-d KD. IHTG was determined by proton magnetic resonance spectroscopy (1H-MRS). The KD diet decreased IHTG by 31% in the face of a 3% decrease in body weight and decreased hepatic insulin resistance (-58%) despite an increase in NEFA concentrations (+35%). These changes were attributed to increased net hydrolysis of IHTG and partitioning of the resulting fatty acids toward ketogenesis (+232%) due to reductions in serum insulin concentrations (-53%) and hepatic citrate synthase flux (-38%), respectively. The former was attributed to decreased hepatic insulin resistance and the latter to increased hepatic mitochondrial redox state (+167%) and decreased plasma leptin (-45%) and triiodothyronine (-21%) concentrations. These data demonstrate heretofore undescribed adaptations underlying the reversal of NAFLD by KD: That is, markedly altered hepatic mitochondrial fluxes and redox state to promote ketogenesis rather than synthesis of IHTG.

Impaired polymorphonuclear leucocyte function in patients undergoing hepatectomy: Adenylate energy charge and superoxide anion production in relation to hepatic mitochondrial redox state

Patients undergoing hepatectomy have an increased susceptibility to infection. We therefore studied the energy metabolism of the polymorphonuclear leucocyte (PMN), focusing on energy charge and function, especially superoxide anion (O2-) generation, in relation to the hepatic mitochondrial redox state. By labelling the PMN adenine nucleotide pool with radioactive adenine and by superoxide dismutase-inhibitable reduction of ferricytochrome c, the energy charge and O2- production was measured in 18 patients with hepatoma (non-cirrhotic, seven; cirrhotic, 11) undergoing hepatectomy. Their arterial ketone body ratios (KBRs), reflecting the hepatic mitochondrial redox potential, were above 0.7 before operation. After surgery, the 18 patients were divided into two groups: group A, KBR greater than 0.7, n = 10; and group B, KBR less than 0.7, n = 8. The energy charge and O2- release in group B decreased significantly from preoperative values (P less than 0.001 and P less than 0.01 respectively) and when compared with group A (P less than 0.05 and P less than 0.01 respectively). These results suggest that impaired hepatic energy metabolism (KBR less than 0.7) in hepatectomized patients leads to impaired energy charge and O2- production in the PMNs.

Alterations in hepatic microcirculation, mitochondrial redox state and tissue oxygenation after trauma and sepsis

Introduction: Hepatic microcirculatory dysfunction and mismatch of blood supply to metabolic demand play an important role in liver failure after stress. Here, we assessed hepatic microcirculation in response to the vasoregulator Endothelin-1 (ET-1) after femur fracture (FFx) and cecal ligation and puncture (CLP). We hypothesized that sequential stress causes hepatic microcirculatory dysfunction leading to altered mitochondrial redox state and tissue PO2 (tPO2). Methods: Male Sprague-Dawley rats (200–300 g, n = 4/groups) underwent sham surgery, FFx, CLP and FFx+CLP. Using intravital microscopy, hepatic microcirculation was assessed by sinusoidal volumetric flow (pL/sec) and perfusion index (PI, pL/sec/200μm). Changes in hepatic mitochondrial redox state and liver tPO2 were determined by NADH autofluorescence (AU, arbitrary unit) and ruthenium (Ru, AU) fluorescence, respectively. Western blot analysis was used to determine ETA/ETB receptor protein expression (AU). Differences among groups were compared using one-way ANOVA followed by Student-Newman-Keuls post hoc test. Results: After 10 minutes of intraportal ET-1 infusion, liver microcirculation was altered in CLP and FFx+CLP groups (Table, values are mean ± SE). TABLE—ABSTRACT 24GroupFlowPINADHRuETAETBSham21 ± 3160 ± 19109 ± 7177 ± 50 ± 09 ± 3FFx19 ± 1155 ± 15101 ± 10148 ± 80 ± 038 ± 8CLP9 ± 2∗69 ± 14∗153 ± 10∗190 ± 6∗0 ± 036 ± 16FFx+CLP5 ± 0.8∗28 ± 4∗160 ± 12∗202 ± 7∗52 ± 8§58 ± 12§∗p < 0.05 vs. sham and FFx,§p < 0.05 vs. sham, FFx, and CLP. Sequential stress also led to increased NADH and Ruthenium fluorescence intensity, indicating a reduction in O2 availability. Vasoactive receptor proteins ETA and ETB were upregulated after injury. Conclusion: Our data suggest that sepsis and sequential stress result in liver microcirculatory disturbances leading to altered global hepatic perfusion, and contribute toward a reduction in O2 supply. These changes in the liver microcirculation, mediated in part by Endothelin-1, may play an important role in the development of liver failure after inflammatory stress.

The serum interleukin 8 level reflects hepatic mitochondrial redox state in hyperthermochemohypoxic isolated liver perfusion with use of a venovenous bypass

Background: We have recently developed a simple method of hyperthermochemohypoxic isolated liver perfusion (HILP) as a regional therapy for unrecognized liver micrometastases. However, little is known about the influence of HILP on cytokine production and liver function. We investigated the influence of HILP on interleukin 8 (IL-8) production and the hepatic mitochondrial function and assessed the relationship between these 2 parameters. We also measured the serum tumor necrosis factor-α (TNF-α) and interleukin 1β (IL-1β) levels to examine the involvement of HILP-induced cytokines in the tumor response. Methods: Sixteen patients with metastatic liver tumors were randomly assigned to undergo hepatectomy with HILP (group A, n = 9) or hepatectomy alone (group B, n = 7). The isolated liver was perfused for 30 minutes with Ringer's lactate solution containing chemotherapeutic agents warmed to 42°C to 43°C without oxygenation. Results: The serum IL-8 levels in group A were markedly increased, with peaks at 3 hours after reperfusion, which was significantly higher than levels in group B (P < .01). In group A the arterial ketone body ratio, which reflects the hepatic mitochondrial redox state, decreased during perfusion and was gradually restored to the preperfusion level 1 hour after reperfusion. However, in group B it decreased during hepatectomy but rapidly recovered 5 minutes after hepatectomy. There was a significant negative correlation between the peak serum IL-8 level and the initial velocity of arterial ketone body ratio recovery for the first 5 minutes after reperfusion r = –0.83, P < .001). The serum TNF-α and IL-1β were temporarily detected only in 3 of 9 patients in group A. Conclusions: We have shown that HILP resulted in augmented IL-8 release but not TNF-α and IL-1β and that the serum IL-8 level reflects the hepatic mitochondrial redox state. These findings suggest that IL-8 production may be associated with hepatic mitochondrial impairment during ischemia. This work may contribute to new therapeutic strategies not only for hepatic ischemia reperfusion injury but also for metastatic liver tumors. (Surgery 1999;125:304-14.)

The serum interleukin 8 level reflects hepatic mitochondrial redox state in hyperthermochemohypoxic isolated liver perfusion with use of a venovenous bypass

Background: We have recently developed a simple method of hyperthermochemohypoxic isolated liver perfusion (HILP) as a regional therapy for unrecognized liver micrometastases. However, little is known about the influence of HILP on cytokine production and liver function. We investigated the influence of HILP on interleukin 8 (IL-8) production and the hepatic mitochondrial function and assessed the relationship between these 2 parameters. We also measured the serum tumor necrosis factor-α (TNF-α) and interleukin 1β (IL-1β) levels to examine the involvement of HILP-induced cytokines in the tumor response. Methods: Sixteen patients with metastatic liver tumors were randomly assigned to undergo hepatectomy with HILP (group A, n = 9) or hepatectomy alone (group B, n = 7). The isolated liver was perfused for 30 minutes with Ringer's lactate solution containing chemotherapeutic agents warmed to 42°C to 43°C without oxygenation. Results: The serum IL-8 levels in group A were markedly increased, with peaks at 3 hours after reperfusion, which was significantly higher than levels in group B ( P < .01). In group A the arterial ketone body ratio, which reflects the hepatic mitochondrial redox state, decreased during perfusion and was gradually restored to the preperfusion level 1 hour after reperfusion. However, in group B it decreased during hepatectomy but rapidly recovered 5 minutes after hepatectomy. There was a significant negative correlation between the peak serum IL-8 level and the initial velocity of arterial ketone body ratio recovery for the first 5 minutes after reperfusion r = –0.83, P < .001). The serum TNF-α and IL-1β were temporarily detected only in 3 of 9 patients in group A. Conclusions: We have shown that HILP resulted in augmented IL-8 release but not TNF-α and IL-1β and that the serum IL-8 level reflects the hepatic mitochondrial redox state. These findings suggest that IL-8 production may be associated with hepatic mitochondrial impairment during ischemia. This work may contribute to new therapeutic strategies not only for hepatic ischemia reperfusion injury but also for metastatic liver tumors. (Surgery 1999;125:304-14.)

Difference in Onset of Warm Ischemia and Reperfusion Injury between Parenchymal and Endothelial Cells of the Liver. Evaluation by Purine Nucleoside Phosphorylase and Hyaluronic Acid

The onset of warm ischemia and reperfusion injury in the liver was investigated in a canine model through changes in parenchymal markers [isozyme class V of lactate dehydrogenase (LDH) and alanine aminotransferase (ALT)], endothelial markers [purine nucleoside phosphorylase (PNP) and hyaluronic acid clearance], and the liver metabolism (ketone body ratio) in warm ischemia induced by inflow occlusion using Pringle's maneuver and subsequent reperfusion. In thisin vivomodel, a PNP assay system and a model were designed so as to exclude the influence of wide localization of PNP possibly originating in erythrocytes or the intestine, and to discriminate between PNP of endothelial cells and that of parenchymal cells in the liver. After 45 min of warm ischemia, reperfusion resulted in damage only to endothelial cells, as seen by significant increase in PNP alone (3.6 ± 0.1 U/liter at the end of warm ischemia to 6.8 ± 0.5 U/liter at 5 min after reperfusion,P< 0.01) and significant decrease in hyaluronic acid clearance compared to the 30-min warm ischemia group in which no increase in either marker for parenchymal and endothelial cells was noted. By contrast, after 60 min of warm ischemia, reperfusion resulted in damage to parenchymal cells along with damage to endothelial cells, as seen by significant increases in LDH(V) and ALT (93 ± 4 U/liter and 32 ± 2 IU/liter at the end of warm ischemia to 239 ± 17 U/liter and 165 ± 27 IU/liter at 5 min after reperfusion, respectively), as well as a marked increase in PNP and deterioration of hyaluronic acid clearance compared to the 45-min warm ischemia group. Reperfusion after 120 min of warm ischemia did not show recovery of metabolic function of the liver as evaluated by hepatic mitochondrial redox state. It is suggested that a time lag occurs in the onset of injury between parenchymal cells and endothelial cells and that endothelial cells are temporally earlier in failing than parenchymal cells when the liver is exposed to short-term warm ischemia and subsequent reperfusion.

Influence of hepatic mitochondrial redox state on complement biosynthesis and activation during and after cardiopulmonary bypass operations.

We have proposed the hazardous phenomena associated with cardiopulmonary bypass (CPB) are due to metabolic derangement by hepatic mitochondrial dysfunction during and after CPB. On the contrary, complement activation and consumption during CPB is reported to be related to the morbidity associated with cardiac surgery. To determine the significance of the hepatic mitochondrial function on the morbidity of cardiac surgery, we measured the serum levels of complements (C3 and C4), activated complements (C3a and C4a), and the arterial ketone body ratio (AKBR), which reflects the hepatic mitochondrial redox state, in 30 patients undergoing CPB. The AKBR, which was at a normal level preoperatively, dropped to a critical level after the initiation of CPB and remained at a low level during the CPB, returning to the preoperative level on the second postoperative morning in a time dependent fashion. The patients group were assigned to two groups according to their AKBR on the first postoperative morning. Group I consisted of patients whose AKBR had recovered to above 0.7 on the first postoperative morning (n = 16). Group II consisted of the rest of the patients (n = 14). The serum complement concentration had considerably decreased by the end of bypass, but recovered in a time-dependent fashion after CPB. The group I patients (C3: 71% of its preoperative value, C4: 85% of its preoperative value) recovered their complements more quickly than the group II patients (C3: 56% of its preoperative value, C4: 54% of its preoperative value). However, the serum C3a and C4a concentrations increased by the end of bypass (C3a: 806% of its preoperative value, C4a: 341% of its preoperative value). The activated complements were significantly higher in the group II patients (C3a: 124% of its preoperative value, C4a: 236% of its preoperative value) than in the group I patients (C3a: 75% of its preoperative value, C4a: 113% of its preoperative value) on the first postoperative morning. It is suggested that hepatic mitochondrial function is related to recovering the complements and to reducing the activated complements after CPB.

Significance of the hepatic mitochondrial redox state in the development of posttraumatic jaundice.

The effect of the hepatic energy status on the development of posttraumatic jaundice (PTJ) was studied to clarify the mechanism of PTJ. Fifty-four patients with severe torso injury who were expected to develop PTJ on admission with an average Injury Severity Score of 27 were selected for this study. They were retrospectively divided into three groups according to their maximum bilirubin concentration by day 10: group H, 12 patients with marked elevation of serum bilirubin (> 8 g/dl); group L, 23 with mild bilirubinemia (2-8 g/dl); and group N, 19 with no bilirubinemia (< 2 g/dl). Group H patients, in whom trauma-related shock was severe and prolonged, developed severe hyperbilirubinemia, and their arterial ketone body ratio (AKBR), which reflects the hepatic mitochondrial redox state and is closely correlated to its energy production, was significantly lower throughout the first week. In contrast, the AKBR increased to an above normal level, indicating enhanced energy production in groups N and L. The serum direct/total bilirubin was also higher in group H. The abnormal hepatic energy metabolism is considered to have reduced the excretion of conjugated bilirubin from the hepatocytes into the bile canaliculi, which is a process that has to proceed against the bilirubin concentration gradient. The subsequent diffusion of the accumulated water-soluble conjugated bilirubin in hepatocytes into the blood is thus considered to be one of the causes of PTJ.

Effect of Growth Hormone on Hepatic Energy Metabolism in Normal Rabbit Liver

Growth hormone (GH), which is well known as an anabolic agent in systemic protein metabolism but has catabolic effects on the carbohydrate metabolism in the liver, was administered to normal rabbit to investigate its effects on the hepatic energy metabolism. The changes in arterial ketone body ratio (AKBR: acetoacetate/3-hydroxybutyrate), which reflects the hepatic mitochondrial redox state ([NAD+]/[NADH]), after GH injection was studied as an indicator of the hepatic energy metabolism. GH was administered to normal rabbit at the doses of 50 micrograms/kg (GH-50 group), 100 micrograms/kg (GH-100 group) and 200 micrograms/kg (GH-200 group) by intravenous bolus injection. In the GH-50, GH-100 and GH-200 groups, AKBR decreased significantly from 1.40 +/- 0.09 to 0.94 +/- 0.05, from 1.19 +/- 0.11 to 0.83 +/- 0.14, and from 1.19 +/- 0.08 to 0.71 +/- 0.15 at 90 min, respectively. The energy charge of the liver decreased significantly 90 min after 200 micrograms/kg GH injection from 0.872 +/- 0.003 to 0.836 +/- 0.012 (p < 0.05). These results suggest that GH is associated with the deterioration of the hepatic energy metabolism, and that the administration of GH should be carefully weighed up in cases of damaged liver.

A new experimental model of specific liver hypoxia using membrane oxygenator.

The present study introduces a new experimental canine model of hepatic arterial deoxygenation using a membrane oxygenator to investigate the influence of hepatic arterial hypoxia on hepatic hemodynamics and energy metabolism. Eighteen mongrel dogs weighing 10 kg each were randomly divided into three groups: group A served as a control (118.0 +/- 9.0 mmHg of hepatic arterial O2 content), group B as a moderately deoxygenated group (40 mmHg of hepatic arterial O2 content), and group C as a severely deoxygenated group (25 mmHg of hepatic arterial O2 content). Deoxygenation was achieved by perfusion of a gas mixture of O2 and N2 through the membrane oxygenator, which was interposed between the femoral artery and the proper hepatic artery, for 60 min. In group C, hypoxia decreased the mean systemic arterial blood pressure and hepatic arterial blood flow. Arterial blood ketone body ratio (AKBR = acetoacetate/3-hydroxybutyrate), which reflects the hepatic mitochondrial redox state, rapidly decreased prior to the significant increase of glutamate oxaloacetate transminase, glutamate pyruvate transminase, and lactate dehydrogenase after the initiation of hypoxia. Hepatic arterial deoxygenation to 25 mmHg for 60 min induced injury to hepatic hemodynamics, resulting in the deterioration of systemic hemodynamics even after the termination of liver hypoxia. This in vivo temporal hepatic arterial hypoxic model without alteration of inflow volume might be useful for investigating the mechanism of hypoxic injury and the critical point of liver hypoxia on hepatic and/or systemic hemodynamics and liver viability.

Functional deterioration of the liver by elevated inferior vena cava pressure: a proposed upper safety limit of pressure for maintaining liver viability in dogs.

Abnormally elevated central venous pressure is considered to be an etiological factor in the onset of acute hepatic failure following modified Fontan operation. This paper hypothesises that an increase in inferior vena cava pressure (IVCP) after such an operation has adverse effects on hepatic energy status. Various degrees of venous hypertension were produced in 10 mongrel dogs by clamping the thoracic IVC with an active veno-venous shunt and varying its flow rate from 60-2.5 ml/min/kg. Arterial ketone body ratio (KBR), reflecting the hepatic mitochondrial redox state, was measured as an index of hepatic energy status. The lower the flow rates of the shunt, the higher the pressures of IVC and portal vein, while systolic blood pressure was maintained above 100 mmHg. CO significantly decreased when the pump speed was less than 30 ml/min/kg. KBR showed a negative correlation to IVCP as well as a positive correlation to portal blood flow (p < 0.05). From the simple regression line obtained between IVCP and KBR, it was determined that an upper safety limit of IVCP may lie at about 27 cmH2O (20.5 mmHg), and that a IVCP of 35 cmH2O (26.6 mmHg) seems to be the critical level for maintaining liver viability.

Redox state in liver mitochondria in acute copper sulfate poisoning

A patient with acute copper sulfate poisoning was found to have maintained a relatively oxidized hepatic mitochondrial redox state in spite of his being in refractory shock. The mechanism underlying this unexpected clinical observation was investigated. The study involved the allocation of male Wistar rats into three groups; copper sulfate-treated (intraperitoneal injection), hemorrhagic shock, and control. Since the copper sulfate-treated rats developed severe hypovolemic shock, hypovolemia was induced in the hemorrhagic shock group to mimic the time course of blood pressure reduction in the copper sulfate-treated group. The control rats were injected intraperitoneally with a saline solution. The hepatic energy charge and the arterial ketone body ratio (AKBR), which reflects the hepatic mitochondrial redox state, of each group of rats was determined. The hepatic energy charge of the copper sulfate-treated rats decreased significantly, reflecting severe hypotension and copper sulfate-induced hepatic damage. This decrease was greater than that observed in the rats subjected to hemorrhagic shock. Despite the profound shock and the markedly decreased hepatic energy charge, the AKBR in copper sulfate-treated rats did not decrease compared with the control group, while the AKBR in rats with induced hemorrhagic shock did decrease significantly. These observations, together with in vitro studies, suggest that the relatively oxidized redox state of liver mitochondria in the copper sulfate-treated rats, notwithstanding the severe shock state of these animals, may be the result of direct oxidation of NADH by copper sulfate.

Apparent normobasemia as a negative prognostic sign immediately after hepatectomy.

The acid-base balance from the postoperative day (POD) 1 to POD 3 after cirrhotic liver resection was investigated in relation to operative outcome by conducting a retrospective study on the changes in arterial ketone body ratio (AKBR), which reflects the hepatic mitochondrial redox state, and arterial blood gas analysis. Patients were grouped according to their outcome: hospital death (HD) and or good outcome, the latter group acting as controls (CTR). Metabolic alkalosis developed in a significant number of the CTR patients (p < 0.01), who had high AKBR values. By contrast, metabolic alkalosis did not develop in the HD group, with significantly lower AKBR (p < 0.001), and there was almost normobasemia. There were no significant differences in the need for mechanical respiratory assistance, the amounts of fresh frozen plasma administered, and renal function test results between the two groups on each of 3 postoperative days. These data indicated that the absence of metabolic alkalosis during the early postoperative days reflects disruption of energy metabolism in the remnant liver, and that the apparent normobasemia is a significant indicator of poor prognosis after cirrhotic liver resection.

Preserved mitochondrial function by allopurinol despite deteriorated hemodynamics in warm ischemia-damaged canine liver

To investigate the pathophysiology of warm ischemia (WI) of the liver, the changes in hemodynamics and energy metabolism were studied during and after 60-min complete WI induced by total hepatic vascular exclusion (HVE) in the canine model. Hepatic arterial blood flow after WI was maintained at 76% of the pre-ischemic level, while portal blood flow was only 27% of the pre-ischemic level associated with increased portal vein pressure, which was twice the pre-ischemic level, resulting in a decrease of total hepatic blood flow to 46% of the pre-ischemic level. Concentration of tissue lipid peroxide increased after WI. Arterial blood ketone body ratio (AKBR), which reflects the hepatic mitochondrial redox state, could not recover to the pre-ischemic level after termination of WI. However, when 100 mg/kg of allopurinol (xanthine oxidase inhibitor) was administered intravenously 10 min prior to initiating WI, AKBR was restored to the pre-ischemic level at 30 min after WI in spite of the fact that allopurinol administration to one group produced no remarkable changes in the hepatic hemodynamics compared with the group without allopurinol treatment. Concentration of adenine nucleotides was significantly higher for the treated group at the end of and after WI than for the group without allopurinol treatment and was maintained at a higher level even after WI. Lipid peroxide production was suppressed. Electron microscopic examination revealed that allopurinol treatment could not prevent mitochondrial swelling. It is suggested that WI causes injury primarily to the portal sinusoidal circulation, resulting in portal congestion concomitant with high portal pressure after the release of WI. Allopurinol could prevent the deterioration of mitochondrial ATP metabolism, and was able to inhibit lipid peroxide production, resulting in the rapid recovery of mitochondrial redox state in spite of the fact that it produced no amelioration of hepatic hemodynamics and morphological alterations.

Simultaneous liver and lung injury following gut ischemia is mediated by xanthine oxidase.

We have previously shown that gut ischemia/reperfusion (I/R) causes simultaneous liver and lung dysfunction and that neutrophils play a critical role in this process. The purpose of this study was to ascertain whether xanthine oxidase (XO) was likewise operational. Normal and XO-inactivated rats (given a tungsten-enriched, molybdenum-depleted diet for 3 weeks) underwent 45 minutes of occlusion of the superior mesenteric artery, and control rats were subjected to a sham laparotomy. After zero and six hours of reperfusion, blood was sampled and livers and lungs harvested. Iodine-125-labeled albumin leak was used as a marker for pulmonary and liver capillary permeability barrier function, and serum acetoacetate/3-hydroxybutyrate (AcAc/3-OHB) levels as an index of hepatic mitochondrial redox state. Gut ischemia/six hours of reperfusion (I/R) increased the 125I albumin lung/blood ratio and the 125I albumin liver/blood ratio; AcAc/3-OHB levels decreased significantly. Xanthine oxidase activation eliminated the observed lung and liver capillary leak as well as the hepatic metabolic derangement induced by gut I/R. In conclusion, the simultaneous lung and liver dysfunction produced by gut I/R is mediated by XO.

Hepatic dysoxia commences during O2 supply dependence.

Hepatic O2 consumption (VO2) remains relatively constant (O2 supply independent) as O2 delivery (DO2) progressively decreases, until a critical DO2 (DO2c) is reached below which hepatic VO2 also decreases (O2 supply dependence). Whether this decrease in VO2 represents an adaptive reduction in O2 demand or a manifestation of tissue dysoxia, i.e., O2 supply that is inadequate to support O2 demand, is unknown. We tested the hypothesis that the decrease in hepatic VO2 during O2 supply dependence represents dysoxia by evaluating hepatic mitochondrial NAD redox state during O2 supply independence and dependence induced by progressive hemorrhage in six pentobarbital-anesthetized dogs. Hepatic mitochondrial NAD redox state was estimated by measuring hepatic venous beta-hydroxybutyrate-to-acetoacetate ratio (beta OHB/AcAc). The value of DO2c was 5.02 +/- 1.64 (SD) ml.100 g-1.min-1. The beta-hydroxybutyrate-to-acetoacetate ratio was constant until a DO2 value (3.03 +/- 1.08 ml.100 g-1.min-1) was reached (P = 0.05 vs. DO2c) and then increased linearly. Peak liver lactate extraction ratio was 15.2 +/- 14.1%, occurring at a DO2 of 5.48 +/- 2.54 ml.100 g-1.min-1 (P = NS vs. DO2c). Our data support the hypothesis that the decrease in VO2 during O2 supply dependence represents tissue dysoxia.

Linear correlation between acetoacetate / β-hydroxybutyrate in arterial bllod and oxidized flavoprotein / reduced pyridine nucleotide in freeze-trapped human liver tissue

A comparison of two methods of measuring liver mitochondrial redox statte demonstrated that a linear correlation exists between acetoacetate / β-hydroxybutyrate ratio in arterial blood (arterial ketone body ratio; AKBR) and oxidized flavoprotein / reduced pyridine nucleotide in human liver tissue (FP/ PN) as measured by tissue fluorescence spectroscopy, such that [FP/PN] = 0.64 + 0.49 × [AKBR] ( r = 0.84, P < 0.001). This result supports the validity of AKBR as a method of measuring the hepatic mitochondrial redox state of pyridine nucleotide using arterial blood.

Liver injury is a reversible neutrophil-mediated event following gut ischemia.

The purposes of this study were to characterize the temporal relationship of distant organ dysfunction after mesenteric ischemia/reperfusion (I/R), and to ascertain if the neutrophil is critical to this process. Normal and neutrophil-depleted rats (vinblastine sulfate, 0.75 mg/kg intravenously) underwent 45 minutes of superior mesenteric artery occlusion and after 0, 6, and 18 hours of reperfusion, blood was sampled and liver and lungs were harvested. Iodine 125 albumin leak was used as a marker for pulmonary and liver injury, and serum acetoacetate/3-hydroxybutyrate (AcAc/3-OHB) was used as an index of hepatic mitochondrial redox state. Gut I/R at 6 hours increased 125I-albumin lung/blood ratio (gut I/R, 0.077 +/- 0.006; control, 0.045 +/- 0.004) and 125I-albumin liver/blood ratio (gut I/R, 0.120 +/- 0.007; control, 0.077 +/- 0.003), while AcAc/3-OHB decreased significantly (gut I/R, 0.420 +/- 0.040; control, 0.880 +/- 0.120). Neutrophil depletion eliminated these changes at 6 hours (blood AcAc/3-OHB, 0.720 +/- 0.100; 125I liver/blood, 0.068 +/- 0.006; 125I lung/blood, 0.046 +/- 0.007). We conclude the following: (1) intestinal I/R produces simultaneous liver and lung injury; (2) injury was present at 6 hours but is reversed at 18 hours; and (3) the I/R-induced liver and lung injuries were neutrophil mediated.