József Kaszaki

Hypoxia-Induced Generation of Methane in Mitochondria and Eukaryotic Cells - An Alternative Approach to Methanogenesis

Background/Aims: Electrophilic methyl groups bound to positively charged nitrogen moieties may act as electron acceptors, and this mechanism could lead to the generation of methane from choline. The aims were to characterize the methanogenic potential of phosphatidylcholine metabolites, and to define the in vivo relevance of this pathway in hypoxia-induced cellular responses. Methods: The postulated reaction was investigated (1) in model chemical experiments, (2) in rat mitochondrial subfractions and (3) in bovine endothelial cell cultures under hypoxic conditions and in the presence of hydroxyl radical generation. The rate of methane formation was determined by gas chromatography with flame-ionisation detectors. The lucigenin-enhanced chemiluminescence assay was used to determine the reactive oxygen species-scavenging capacity of the choline metabolites. Results: Significant methane generation was demonstrated in all three series of experiments. Phosphatidylcholine metabolites with alcoholic moiety in the molecule (i.e. choline, N,N-dimethylethanolamine and N-methylethanolamine), inhibited oxygen radical production both in vitro and in vivo, and displayed an effectiveness proportional to the amount of methane generated and the number of methyl groups in the compounds. Conclusion: Methane generation occurs in aerobic systems. Phosphatidylcholine metabolites containing both electron donor and acceptor groups may have a function to counteract intracellular oxygen radical production.

NF-κB activation is detrimental in arginine-induced acute pancreatitis

The transcription factor nuclear factor kappaB (NF-kappaB) has been shown to have a critical role in the pathogenesis of sodium taurocholate- and cerulein-induced acute pancreatitis by regulating the expression of many proinflammatory genes in the pancreas. Heat shock proteins (HSPs), on the other hand, protect the pancreas against cellular damage. The aims of the present study were: (i) to investigate pancreatic NF-kappaB activation, proinflammatory cytokine synthesis, and cytoprotective HSP induction during L-arginine- (Arg-) induced acute pancreatitis in rats, and (ii) to establish whether pretreatment with pyrrolidine dithiocarbamate (PDTC) or methylprednisolone (MP) can block the activation of pancreatic NF-kappaB and determine their effects on the severity of Arg-induced acute pancreatitis. The dose-response (3 or 4 g/kg) and time-effect (0.5-96 h) curves relating to the action of Arg on pancreatic NF-kappaB activation and IL-1beta, TNF-alpha, HSP60, and HSP72 synthesis were evaluated. Various doses of PDTC or MP were administered 1 h before the induction of pancreatitis. We demonstrated that Arg specifically and dose-dependently induces pancreatitis, activates NF-kappaB (only the 3 g/kg dose) and proinflammatory cytokine synthesis, and increases the expressions of HSP60 and HSP72 in the pancreas of rats. The lower dose of Arg induced a less severe pancreatitis, but larger increases in the levels of HSPs. The present work supports and extends earlier observations that NF-kappaB activation is a common mechanism in acute pancreatitis, although it is dose dependent and occurs at a later stage in Arg-induced pancreatitis as compared with other models. PDTC and MP pretreatment dose-dependently blocked NF-kappaB activation and proinflammatory cytokine expression and ameliorated many of the examined laboratory (the pancreatic weight/body weight ratio, the pancreatic myeloperoxidase activity, the pancreatic contents of protein, amylase and trypsinogen, the degrees of lipid peroxidation and protein oxidation, and the nonprotein sulfhydryl group content) and morphological parameters of the disease. These findings suggest that pretreatment with PDTC or MP has an anti-inflammatory effect during Arg-induced pancreatitis, which is at least partly mediated by the inhibition of NF-kappaB activation and proinflammatory cytokine synthesis. The increased levels of HSPs most probably act to limit the severity of the disease.

The anti-inflammatory effects of methane.

Objective:Gastrointestinal methane generation has been demonstrated in various stress conditions, but it is not known whether nonasphyxiating amounts have any impact on the mammalian pathophysiology. We set out to characterize the effects of exogenous methane administration on the process of inflammatory events arising after reoxygenation in a large animal model of ischemia–reperfusion. Design:A randomized, controlled in vivo animal study. Setting:A university research laboratory. Subjects:Inbred beagle dogs (12.7 6 2 kg). Interventions:Sodium pentobarbital-anesthetized animals were randomly assigned to sham-operated or ischemia–reperfusion groups, where superior mesenteric artery occlusion was maintained for 1 hr and the subsequent reperfusion was monitored for 3 hrs. For 5 mins before reperfusion, the animals were mechanically ventilated with normoxic artificial air with or without 2.5% methane. Biological responses to methane–oxygen respirations were defined in pilot rat studies and assay systems were used with xanthine oxidase and activated canine granulocytes to test the in vitro bioactivity potential of different gas concentrations. Measurements and Main Results:The macrohemodynamics and small intestinal pCO2 gap changes were recorded and peripheral blood samples were taken for plasma nitrite/nitrate and myeloperoxidase analyses. Tissue superoxide and nitrotyrosine levels and myeloperoxidase activity changes were determined in intestinal biopsy samples; structural mucosal damage was measured by hematoxylin and eosin staining. Methane inhalation did not influence the macrohemodynamics but significantly reduced the magnitude of the tissue damage and the intestinal pCO2 gap changes after reperfusion. Furthermore, the plasma and mucosal myeloperoxidase activity and the intestinal superoxide and nitrotyrosine levels were reduced, whereas the plasma nitrite/nitrate concentrations were increased. Additionally, methane effectively and specifically inhibited leukocyte activation in vitro. Conclusions:These data demonstrate the anti-inflammatory profile of methane. The study provides evidence that exogenous methane modulates leukocyte activation and affects key events of ischemia–reperfusion-induced oxidative and nitrosative stress and is therefore of potential therapeutic interest in inflammatory pathologies. (Crit Care Med 2012; 40:–1278)

B-lines quantify the lung water content: A lung ultrasound versus lung gravimetry study in acute lung injury

B-lines (also termed ultrasound lung comets) obtained with lung ultrasound detect experimental acute lung injury (ALI) very early and before hemogasanalytic changes, with a simple, noninvasive, nonionizing and real-time method. Our aim was to estimate the correlation between B-lines number and the wet/dry ratio of the lung tissue, measured by gravimetry, in an experimental model of ALI. Seventeen Na-pentobarbital anesthetized, cannulated (central vein and carotid artery) minipigs were studied: five sham-operated animals served as controls and, in 12 animals, ALI was induced by injection of oleic acid (0.1 mL/kg) via the central venous catheter. B-lines were measured by echographic scanner in four predetermined chest scanning sites in each animal. At the end of each experiment, both lungs were dissected, weighed and dried to determine wet/dry weight ratio by gravimetry. After the injection of oleic acid, B-lines number increased over time. A significant correlation was found between the wet/dry ratio and B-lines number (r = 0.91, p < 0.001). These data suggest that in an experimental pig model of ALI/ARDS, B-lines assessed by lung ultrasound provide a simple, semiquantitative, noninvasive index of lung water accumulation, strongly correlated to invasive gravimetric assessment.

Kynurenic acid inhibits intestinal hypermotility and xanthine oxidase activity during experimental colon obstruction in dogs

Abstract  Kynurenic acid (KynA), an endogenous antagonist of N‐methyl‐d‐aspartate (NMDA) glutamate receptors, protects the central nervous system in excitotoxic neurological diseases. We hypothesized that the inhibition of enteric glutamate receptors by KynA may influence dysmotility in the gastrointestinal tract. Group 1 of healthy dogs served as the sham‐operated control, in group 2, the animals were treated with KynA, while in groups 3 and 4 mechanical colon obstruction was maintained for 7 h. Group 4 was treated with KynA at the onset of ileus. Hemodynamics and motility changes were monitored, and the activities of xanthine oxidoreductase (XOR) and myeloperoxidase (MPO) were determined from tissue samples. Colon obstruction induced a hyperdynamic circulatory reaction, significantly elevated the motility index and increased the mucosal leucocyte accumulation and the XOR activity. The KynA treatment augmented the tone of the colon, permanently decreased the motility index of the giant colonic contractions and reduced the increases in XOR and MPO activities. These effects were concomitant with the in vitroinhibition of XOR activity. In conclusion, KynA antagonizes the obstruction‐induced motility responses and XOR activation in the colon. Inhibition of enteric NMDA receptors may provide an option to influence intestinal hypermotility and inflammatory changes.

Oral phosphatidylcholine pretreatment decreases ischemia-reperfusion induced methane generation and the inflammatory response in the small intestine

We have shown that phosphatidylcholine (PC) metabolites may have a function in counteracting the production of reactive oxygen species (ROS), and that this mechanism can lead to the generation of methane from choline. The aims were to establish whether the dietary administration of PC can protect the reperfused small bowel mucosa by its acting as an anti-inflammatory agent and to investigate this possibility in association with in vivo methane generation. Group 1 (n = 5) of anesthetized dogs served as sham-operated controls, whereas in groups 2 (n = 6) and 3 (n = 6), complete small intestinal ischemia was induced by occluding the superior mesenteric artery for 60 min. Groups 1 and 2 were fed with normal laboratory chow for 1 week before the experiments, whereas the animals in group 3 received a special diet containing 1% soybean PC. The intramucosal pH and the difference of the arterial and local PCO2 (PCO2 gap) were detected by indirect tonometry. Intestinal superoxide production and myeloperoxidase (MPO) activity (a marker of tissue leukocyte infiltration) were ascertained on ileal biopsy samples 180 min after reperfusion. The content of methane in the exhaled air was determined by gas chromatography. I/R was characterized by significant tissue acidosis with ROS generation and elevated MPO activity. These changes were accompanied by increased methane production in the exhaled air during reoxygenation. The PC-enriched diet prevented the decrease in intramucosal pH, diminished the intestinal superoxide generation and the MPO activity, and significantly decreased the exhaled methane concentration. The increased dietary uptake of PC exerts an anti-inflammatory influence in the gastrointestinal tract. Exhaled methane is linked to abnormal ROS generation; a decreased methane production is associated with significantly reduced inflammatory activation during I/R.

N-Methyl-D-aspartate receptor antagonism decreases motility and inflammatory activation in the early phase of acute experimental colitis in the rat.

Background  Inflammatory bowel diseases are accompanied by severe motility disorders. The aim of our study was to investigate whether the blockade of peripheral N‐methyl‐D‐aspartate (NMDA)‐sensitive glutamate receptors (NMDA‐Rs) alters motility changes in chemically induced acute colitis and how this modulation is accomplished.

The role of mast cells in mucosal permeability changes during ischemia-reperfusion injury of the small intestine

The objective of this study was to investigate the significance of mast cell-induced reactions in the mucosal functional and morphological alterations induced by 30 min segmental ischemia and 120 min reperfusion in anesthetized dogs. The rates of changes in permeability of the mucosa to sodium fluorescein (NaFL) in the plasma-to-lumen and lumen-to-plasma directions were studied, the local hemodynamics, intramucosal pH (pHi) alterations, mast cell number and degranulation, and degree of tissue injury were determined. The effects of pretreatments with cromolyn (a peritoneal-type mast cell stabilizer), quercetin (a mucosal-type mast cell stabilizer), and dexamethasone (an aspecific membrane stabilizer and mast cell depleter) were evaluated. We found that ischemia-reperfusion induced significant tissue injury, elevated the segmental vascular resistance, and decreased pHi. The plasma-to-lumen clearance of NaFL increased significantly during ischemia and reperfusion. Cromolyn and quercetin pretreatments significantly inhibited the permeability changes, but did not influence the pHi and morphological alterations induced by ischemia-reperfusion. Dexamethasone pretreatment did not influence the number of mast cells, but the degree of mast cell degranulation and fluorescein leakage decreased. We conclude that intestinal mast cells and mast cell-induced reactions contribute to the mucosai permeability alterations during reperfusion, but play only a minor role in ischemia-reperfusion-induced structural injury.

A new severe acute necrotizing pancreatitis model induced by l-ornithine in rats

Objective:Intraperitoneal administration of large doses of l-arginine is known to induce severe acute pancreatitis in rats. We therefore set out to determine whether metabolites of l-arginine (l-ornithine, l-citrulline, and nitric oxide) cause pancreatitis. Design:The authors conducted an in vivo animal study. Setting:This study was conducted at a university research laboratory. Subjects:Study subjects were male Wistar rats. Interventions:Dose–response and time course changes of laboratory and histologic parameters of pancreatitis were determined after l-arginine, l-ornithine, l-citrulline, or sodium nitroprusside (nitric oxide donor) injection. Measurements and Main Results:Intraperitoneal injection of 3 g/kg l-ornithine but not l-citrulline or nitroprusside caused severe acute pancreatitis; 4 to 6 g/kg l-ornithine killed the animals within hours. Serum and ascitic amylase activities were significantly increased, whereas pancreatic amylase activity was decreased after intraperitoneal injection of 3 g/kg l-ornithine. The increase in pancreatic trypsin activity (9–48 hrs) correlated with the degradation of I&kgr;B proteins and elevated interleukin-1&bgr; levels. Oxidative stress in the pancreas was evident from 6 hrs; HSP72 synthesis was increased from 4 hrs after l-ornithine administration. Morphologic examination of the pancreas showed massive interstitial edema, apoptosis, and necrosis of acinar cells and infiltration of neutrophil granulocytes and monocytes 18 to 36 hrs after 3 g/kg l-ornithine injection. One month after l-ornithine injection, the pancreas appeared almost normal; the destructed parenchyma was partly replaced by fat. Equimolar administration of l-arginine resulted in lower pancreatic weight/body weight ratio, pancreatic myeloperoxidase activity, and histologic damage compared with the l-ornithine-treated group. l-ornithine levels in the blood were increased 54-fold after intraperitoneal administration of l-arginine. Conclusions:We have developed a simple, noninvasive model of acute necrotizing pancreatitis in rats by intraperitoneal injection of 3 g/kg l-ornithine. Interestingly, we found that, compared with l-arginine, l-ornithine was even more effective at inducing pancreatitis. Large doses of l-arginine produce a toxic effect on the pancreas, at least in part, through l-ornithine.

Zerumbone exerts a beneficial effect on inflammatory parameters of cholecystokinin octapeptide-induced experimental pancreatitis but fails to improve histology.

Objective: Our experiments were designed to investigate the effects of zerumbone pretreatment on cholecystokinin octapeptide (CCK-8)-induced acute pancreatitis in rats. Methods: Male Wistar rats weighing 240 to 280 g were divided into a control group, a group treated with CCK-8, a group receiving 20 mg/kg zerumbone before CCK-8 administration, and a group treated with zerumbone only. Results: The serum amylase and lipase activities and the pancreatic weight-body weight ratio were significantly reduced by zerumbone pretreatment, but the drug failed to influence the histological parameters of pancreatitis. The anti-inflammatory effects of the drug were manifested in decreases in the cytosolic interleukin 6 and tumor necrosis factor &agr; concentrations and an elevation in the I-&kgr;B concentration, whereas the antioxidant ability of zerumbone was demonstrated by reductions in inducible nitric oxide synthase, Mn- and Cu/Zn-superoxide dismutase activities in the zerumbone-treated rats. Conclusion: Zerumbone ameliorated the changes of several parameters of acute pancreatitis probably by interfering with I-&kgr;B degradation, but in the applied dose, it failed to influence the histology of the disease.Abbreviations: CCK-8 - cholecystokinin octapeptide, DMSO - dimethyl sulfoxide, iNOS - inducible nitric oxide synthase, cNOS - constitutive nitric oxide synthase, TNF, tumor necrosis factor, IL-6 - interleukin 6, NF-&kgr;B - nuclear factor &kgr;B, SOD - superoxide dismutase, ASAT - aspartate aminotransferase