Effects of prolonged endotoxemia on liver, skeletal muscle and kidney mitochondrial function

INTRODUCTION: Sepsis may impair mitochondrial utilization of oxygen. Since hepatic dysfunction is a hallmark of sepsis, we hypothesized that the liver is more susceptible to mitochondrial dysfunction than the peripheral tissues, such as the skeletal muscle. We studied the effect of prolonged endotoxin infusion on liver, muscle and kidney mitochondrial respiration and on hepatosplanchnic oxygen transport and microcirculation in pigs. METHODS: Twenty anesthetized pigs were randomized to receive either endotoxin or saline infusion for 24 hours. Muscle, liver and kidney mitochondrial respiration was assessed. The cardiac output (thermodilution) and the carotid, superior mesenteric and kidney arterial, portal venous (ultrasound Doppler) and microcirculatory blood flow (laser Doppler) were measured, and systemic and regional oxygen transport and lactate exchange were calculated. RESULTS: Endotoxin infusion induced hyperdynamic shock and impaired the glutamate-dependent and succinate-dependent mitochondrial respiratory control ratio in the liver (glutamate, median (range) endotoxemia 2.8 (2.3–3.8) vs controls 5.3 (3.8–7.0); P < 0.001; succinate, endotoxemia 2.9 (1.9–4.3) vs controls 3.9 (2.6–6.3), P = 0.003). While the ADP added/oxygen consumed ratio was reduced with both substrates, the maximal ATP production was impaired only in the succinate-dependent respiration. Hepatic oxygen consumption and extraction, and the liver surface laser Doppler blood flow remained unchanged. Glutamate-dependent respiration in the muscle and kidney was unaffected. CONCLUSION: Endotoxemia reduces the efficiency of hepatic mitochondrial respiration but neither skeletal muscle nor kidney mitochondrial respiration, independent of regional and microcirculatory blood flow changes

EFFECT OF METHYLENE BLUE ON THE DISPOSITION OF ETHANOL

The effect of methylene blue on the disposition of ethanol was studied in rats and humans. Methylene blue increased the metabolism of [14C]ethanol to 14CO2 in isolated hepatocytes and in intact rats by 75% and 30%, respectively. In healthy volunteers, methylene blue did not affect the pharmacokinetics of ethanol and did not alleviate the ethanol-induced NAD redox changes as reflected by the increase in the [lactate]/[pyruvate] rati

Rapid determination of gemcitabine in plasma and serum using reversed-phase HPLC

Gemcitabine (2'2'-difluorodeoxycytidine) is a pyrimidine analog used in the treatment of a variety of solid tumors. After intravenous (i.v.) administration, it is rapidly inactivated to 2'-deoxy-2',2'-difluorouridine (dFdU). A sensitive analytical method for the quantitation of gemcitabine is required for the assessment of alternative dosage and treatment schemes. A rapid and robust RP-HPLC assay for analysis of gemcitabine in human and animal plasma and serum was developed and validated using 2'-deoxyuridine (dU) and 5-fluoro-2'-deoxyuridine (5FdU) as internal standards. It is based on protein precipitation, the use of an Atlantis dC18 column of 100 mm length (inner diameter, 4.6 mm; particle size, 3 microm) and isocratic elution using a 10 mM phosphate buffer, pH 3.0, followed by isocratic elution with the same buffer containing 3% of ACN. For gemcitabine, RSD values for intraday and interday precision were or =89%. The recovery for gemcitabine, dU and 5FdU was 86-98%. The assay was applied to determine gemcitabine levels in plasma samples of patients collected during and shortly after conventional infusion of 25-30 mg/kg body mass (levels: 2.0-18.9 microg/mL) and rats that received lower doses (1.5 mg/kg) via i.v., subcutaneous and oral drug administration (levels: 0.20-2.60 microg/mL). It could also be applied to estimate dFdU levels in human plasma

Biliary excretion of glutathione and glutathione

Abstract. Regulation of the biliary excretion of reduced glutathione (GSH) and glutathione disulfide (GSSG) and responses to selected model toxins were examined in male Sprague-Dawley rats. In control and phenobarbital-pretreated rats in which the intrahepatic concentration of GSH was modulated by the administration of diethyl maleate or acetaminophen, the biliary concentration of GSH was consistently lower than, but directly proportional to, the intrahepatic concentration of GSH. Furthermore, increments in bile flow produced by the infusion of sulfobromophthalein (BSP)-glutathione were associated with proportional increases in the biliary excretion of GSH, suggesting that GSH passes into bile passively along a concentration gradient. In contrast, GSSG appears to be secreted into bile against a steep concentration gradient. An increased hepatic production and biliary excretion of GSSG resulted from the administration of t-butyl hydroperoxide. Measurement of biliary GSSG and BSP during a constant infusion of the GSH adduct of BSP indicated that GSSG shares a common excretory mechanism with GSH adducts. Diquat, nitrofurantoin, and paraquat also markedly stimulated the biliary excretion of GSSG. On a molar basis, these compounds generated much more GSSG than a direct substrate for glutathione peroxidase such as t-butyl hydroperoxide, indicating that the compounds undergo redox-cycling with concomitant production of hydrogen peroxide. Aminopyrine (0.8 mmol/kg) also significantly increased biliary GSSG. This increase, however, was associated with a proportional increase in bile flow and in the biliary excretion of GSH such that the GSSG/GS

Liver breath tests non-invasively predict higher stages of non-alcoholic steatohepatitis

Effectively assessing subtle hepatic metabolic functions by novel non-invasive tests might be of clinical utility in scoring NAFLD (non-alcoholic fatty liver disease) and in identifying altered metabolic pathways. The present study was conducted on 39 (20 lean and 19 obese) hypertransaminasemic patients with histologically proven NAFLD {ranging from simple steatosis to severe steatohepatitis [NASH (non-alcoholic steatohepatitis)] and fibrosis} and 28 (20 lean and eight overweight) healthy controls, who underwent stable isotope breath testing ([(13)C]methacetin and [(13)C]ketoisocaproate) for microsomal and mitochondrial liver function in relation to histology, serum hyaluronate, as a marker of liver fibrosis, and body size. Compared with healthy subjects and patients with simple steatosis, NASH patients had enhanced methacetin demethylation (P=0.001), but decreased (P=0.001) and delayed (P=0.006) ketoisocaproate decarboxylation, which was inversely related (P=0.001) to the degree of histological fibrosis (r=-0.701), serum hyaluronate (r=-0.644) and body size (r=-0.485). Ketoisocaproate decarboxylation was impaired further in obese patients with NASH, but not in patients with simple steatosis and in overweight controls. NASH and insulin resistance were independently associated with an abnormal ketoisocaproate breath test (P=0.001). The cut-off value of 9.6% cumulative expired (13)CO(2) for ketoisocaproate at 60 min was associated with the highest prediction (positive predictive value, 0.90; negative predictive value, 0.73) for NASH, yielding an overall sensitivity of 68% and specificity of 94%. In conclusion, both microsomal and mitochondrial functions are disturbed in NASH. Therefore stable isotope breath tests may usefully contribute to a better and non-invasive characterization of patients with NAFLD

Low membrane protein sulfhydrils but not G6PD deficiency predict ribavirin-induced hemolysis in hepatitis C

Hemolysis is a frequent adverse effect of ribavirin (RBV). It has been suggested that oxidative stress plays a role, but mechanisms and predictive risk factors for severe forms remain unknown. Markers of redox status were determined in erythrocytes of 34 patients with hepatitis C-four of them with glucose-6-phosphate-dehydrogenase (G6PD) deficiency-before and during treatment with RBV and interferon (IFN) and were compared with 10 healthy control subjects. In addition, erythrocytes were incubated with RBV, and the effects of dipyridamole (DPD), diethylmaleate (DEM), and glutathione ester (GSHE) were studied in vitro. Of the 30 patients without G6PD deficiency who were treated with RBV and IFN-alpha, five developed major hemolysis (Delta hemoglobin &gt; 6 g/dL) and 25 developed minor hemolysis (Delta hemoglobin &lt; 2.5 g/dL). Patients with major hemolysis had lower median pretreatment values of membrane protein sulfhydrils than patients with minor hemolysis (28.4 vs. 36.7 nmol/mg, P &lt;.001). Erythrocytes of G6PD-deficient patients were not more susceptible to RBV-induced hemolysis. In in vitro incubations of erythrocytes, DEM enhanced the RBV-induced decrease of glutathione, protein sulfhydrils, and osmotic resistance. Supplementation of GSHE and DPD prevented the RBV-induced decrease in osmotic resistance, adenosyl triphosphate (ATP), and 2,3-diphosphoglycerate (DPG), the loss of glutathione and protein sulfhydrils, and the formation of thiobarbituric acid reactive substances (TBARs). In conclusion, the data indicate that low membrane protein sulfhydrils prior to therapy but not G6PD deficiency are predictive of RBV-induced major hemolysis. In vitro, GSHE and DPD reduce the RBV-associated oxidative stress in erythrocytes and prevent the increase in osmotic fragility, suggesting that these compounds might decrease the risk of hemolysis in patient