Eszter Tuboly

The role of methane in mammalian physiology - Is it a gasotransmitter?

Mammalian methanogenesis is widely considered to be an exclusive sign of anaerobic microbial activity in the gastrointestinal tract. This commonly held view was challenged, however, when in vitro and in vivo investigations demonstrated the possibility of nonmicrobial methane formation in aerobic organisms, in plants and animals. The aim of this review is to discuss the available literature data on the biological role of methane. When we evaluate the significance of methane generation in the mammalian physiology, the question may be examined: is it a gas mediator? Overall the data do not fully support the gasotransmitter concept, but they do support the notion that methane liberation may be linked to redox regulation and may be connected with hypoxic events leading to, or associated with a mitochondrial dysfunction. In this respect, the available information suggests that hypoxia-induced methane generation may be a necessary phenomenon of aerobic life, and perhaps a surviving evolutionary trait in the eukaryote cell.

Methane biogenesis during sodium azide-induced chemical hypoxia in rats.

Previous studies demonstrated methane generation in aerobic cells. Our aims were to investigate the methanogenic features of sodium azide (NaN(3))-induced chemical hypoxia in the whole animal and to study the effects of l-α-glycerylphosphorylcholine (GPC) on endogenous methane production and inflammatory events as indicators of a NaN(3)-elicited mitochondrial dysfunction. Group 1 of Sprague-Dawley rats served as the sham-operated control; in group 2, the animals were treated with NaN(3) (14 mg·kg(-1)·day(-1) sc) for 8 days. In group 3, the chronic NaN(3) administration was supplemented with daily oral GPC treatment. Group 4 served as an oral antibiotic-treated control (rifaximin, 10 mg·kg(-1)·day(-1)) targeting the intestinal bacterial flora, while group 5 received this antibiotic in parallel with NaN(3) treatment. The whole body methane production of the rats was measured by means of a newly developed method based on photoacoustic spectroscopy, the microcirculation of the liver was observed by intravital videomicroscopy, and structural changes were assessed via in vivo fluorescent confocal laser-scanning microscopy. NaN(3) administration induced a significant inflammatory reaction and methane generation independently of the methanogenic flora. After 8 days, the hepatic microcirculation was disturbed and the ATP content was decreased, without major structural damage. Methane generation, the hepatic microcirculatory changes, and the increased tissue myeloperoxidase and xanthine oxidoreductase activities were reduced by GPC treatment. In conclusion, the results suggest that methane production in mammals is connected with hypoxic events associated with a mitochondrial dysfunction. GPC is protective against the inflammatory consequences of a hypoxic reaction that might involve cellular or mitochondrial methane generation.

Inhaled Methane Limits the Mitochondrial Electron Transport Chain Dysfunction during Experimental Liver Ischemia-Reperfusion Injury

Background Methanogenesis can indicate the fermentation activity of the gastrointestinal anaerobic flora. Methane also has a demonstrated anti-inflammatory potential. We hypothesized that enriched methane inhalation can influence the respiratory activity of the liver mitochondria after an ischemia-reperfusion (IR) challenge. Methods The activity of oxidative phosphorylation system complexes was determined after in vitro methane treatment of intact liver mitochondria. Anesthetized Sprague-Dawley rats subjected to standardized 60-min warm hepatic ischemia inhaled normoxic air (n = 6) or normoxic air containing 2.2% methane, from 50 min of ischemia and throughout the 60-min reperfusion period (n = 6). Measurement data were compared with those on sham-operated animals (n = 6 each). Liver biopsy samples were subjected to high-resolution respirometry; whole-blood superoxide and hydrogen peroxide production was measured; hepatocyte apoptosis was detected with TUNEL staining and in vivo fluorescence laser scanning microscopy. Results Significantly decreased complex II-linked basal respiration was found in the normoxic IR group at 55 min of ischemia and a lower respiratory capacity (~60%) and after 5 min of reperfusion. Methane inhalation preserved the maximal respiratory capacity at 55 min of ischemia and significantly improved the basal respiration during the first 30 min of reperfusion. The IR-induced cytochrome c activity, reactive oxygen species (ROS) production and hepatocyte apoptosis were also significantly reduced. Conclusions The normoxic IR injury was accompanied by significant functional damage of the inner mitochondrial membrane, increased cytochrome c activity, enhanced ROS production and apoptosis. An elevated methane intake confers significant protection against mitochondrial dysfunction and reduces the oxidative damage of the hepatocytes.

Exhaled methane concentration profiles during exercise on an ergometer

Exhaled methane concentration measurements are extensively used in medical investigation of certain gastrointestinal conditions. However, the dynamics of endogenous methane release is largely unknown. Breath methane profiles during ergometer tests were measured by means of a photoacoustic spectroscopy based sensor. Five methane-producing volunteers (with exhaled methane level being at least 1 ppm higher than room air) were measured. The experimental protocol consisted of 5 min rest--15 min pedalling (at a workload of 75 W)--5 min rest. In addition, hemodynamic and respiratory parameters were determined and compared to the estimated alveolar methane concentration. The alveolar breath methane level decreased considerably, by a factor of 3-4 within 1.5 min, while the estimated ventilation-perfusion ratio increased by a factor of 2-3. Mean pre-exercise and exercise methane concentrations were 11.4 ppm (SD:7.3) and 2.8 ppm (SD:1.9), respectively. The changes can be described by the high sensitivity of exhaled methane to ventilation-perfusion ratio and are in line with the Farhi equation.

Peripheral inflammatory activation after hippocampus irradiation in the rat

Abstract Purpose: To detect the possible biochemical signs of inflammatory activation in the peripheral circulation in a rodent model of hippocampus irradiation, and to examine the effects of L-alpha-glycerylphosphorylcholine (GPC) in this experimental protocol. Materials and methods: Anesthetized Sprague-Dawley rats were subjected to 40 Gy cobalt irradiation of both hemispheres of the hippocampus, with or without GPC treatment (50 mg/kg intravenously (i.v.), 5 min before the irradiation, n = 6, each). A third group (n = 6) served as saline-treated control. Blood samples were obtained 3 h after the end of irradiation in order to examine the changes in plasma histamine, tumor necrosis factor-alpha (TNF-α), interleukin 1-beta, interleukin 6 (IL-6) and interleukin 10 (IL-10); liver tissue samples were taken to determine adenosine triphosphate (ATP) concentrations. Results: The hepatic ATP levels were significantly declined, while plasma concentrations of circulating TNF-α, IL-6, IL-10 and histamine were significantly increased after hippocampus irradiation. GPC treatment significantly reduced the irradiation-induced release of cytokines and histamine, and the liver ATP level was maintained at the control value. Conclusions: Targeted brain irradiation produced measurable pro- and anti-inflammatory cytokine changes in the systemic circulation. GPC supplementation provides significant protection against irradiation-induced peripheral pro-inflammatory activation and ATP depletion.

C5a inhibitor protects against ischemia/reperfusion injury in rat small intestine

Acute mesenteric ischemia (AMI) is caused by considerable intestinal injury, which is associated with intestinal ischemia followed by reperfusion. To elucidate the mechanisms of ischemia/reperfusion injuries, a C5a inhibitory peptide termed AcPepA was used to examine the role of C5a anaphylatoxin, induction of inflammatory cells, and cell proliferation of the intestinal epithelial cells in an experimental AMI model. In this rat model, the superior mesenteric artery was occluded and subsequently reperfused (Induce‐I/R). Other groups were treated with AcPepA before ischemia or reperfusion. Induce‐I/R induced injuries in the intestine and AcPepA significantly decreased the proportion of severely injured villi. Induce‐I/R induced secondary receptor for C5a‐positive polymorphonuclear leukocytes in the vessels and CD204‐positive macrophages near the injured site; this was correlated with hypoxia‐induced factor 1‐alpha‐positive cells. Induction of these inflammatory cells was attenuated by AcPepA. In addition, AcPepA increased proliferation of epithelial cells in the villi, possibly preventing further damage. Therefore, Induce‐I/R activates C5a followed by the accumulation of polymorphonuclear leukocyte and hypoxia‐induced factor 1‐alpha‐producing macrophages, leading to villus injury. AcPepA, a C5a inhibitory peptide, blocks the deleterious effects of C5a, indicating it has a therapeutic effect on the inflammatory consequences of experimental AMI.

Inhalation of methane preserves the epithelial barrier during ischemia and reperfusion in the rat small intestine

Background: Methane is part of the gaseous environment of the intestinal lumen. The purpose of this study was to elucidate the bioactivity of exogenous methane on the intestinal barrier function in an antigen‐independent model of acute inflammation. Methods: Anesthetized rats underwent sham operation or 45‐min occlusion of the superior mesenteric artery. A normoxic methane (2.2%)‐air mixture was inhaled for 15 min at the end of ischemia and at the beginning of a 60‐min or 180‐min reperfusion. The integrity of the epithelial barrier of the ileum was assessed by determining the lumen‐to‐blood clearance of fluorescent dextran, while microvascular permeability changes were detected by the Evans blue technique. Tissue levels of superoxide, nitrotyrosine, myeloperoxidase, and endothelin‐1 were measured, the superficial mucosal damage was visualized and quantified, and the serosal microcirculation and mesenteric flow was recorded. Erythrocyte deformability and aggregation were tested in vitro. Results: Reperfusion significantly increased epithelial permeability, worsened macro‐ and microcirculation, increased the production of proinflammatory mediators, and resulted in a rapid loss of the epithelium. Exogenous normoxic methane inhalation maintained the superficial mucosal structure, decreased epithelial permeability, and improved local microcirculation, with a decrease in reactive oxygen and nitrogen species generation. Both the deformability and aggregation of erythrocytes improved with incubation of methane. Conclusion: Normoxic methane decreases the signs of oxidative and nitrosative stress, improves tissue microcirculation, and thus appears to modulate the ischemia‐reperfusion–induced epithelial permeability changes. These findings suggest that the administration of exogenous methane may be a useful strategy for maintaining the integrity of the mucosa sustaining an oxido‐reductive attack.

Excessive alcohol consumption induces methane production in humans and rats

Various studies have established the possibility of non-bacterial methane (CH4) generation in oxido-reductive stress conditions in plants and animals. Increased ethanol input is leading to oxido-reductive imbalance in eukaryotes, thus our aim was to provide evidence for the possibility of ethanol-induced methanogenesis in non-CH4 producer humans, and to corroborate the in vivo relevance of this pathway in rodents. Healthy volunteers consumed 1.15 g/kg/day alcohol for 4 days and the amount of exhaled CH4 was recorded by high sensitivity photoacoustic spectroscopy. Additionally, Sprague-Dawley rats were allocated into control, 1.15 g/kg/day and 2.7 g/kg/day ethanol-consuming groups to detect the whole-body CH4 emissions and mitochondrial functions in liver and hippocampus samples with high-resolution respirometry. Mitochondria-targeted L-alpha-glycerylphosphorylcholine (GPC) can increase tolerance to liver injury, thus the effects of GPC supplementations were tested in further ethanol-fed groups. Alcohol consumption was accompanied by significant CH4 emissions in both human and rat series of experiments. 2.7 g/kg/day ethanol feeding reduced the oxidative phosphorylation capacity of rat liver mitochondria, while GPC significantly decreased the alcohol-induced CH4 formation and hepatic mitochondrial dysfunction as well. These data demonstrate a potential for ethanol to influence human methanogenesis, and suggest a biomarker role for exhaled CH4 in association with mitochondrial dysfunction.

C5a inhibitor protects ischemia/reperfusion injury in rat small intestine

Acute mesenteric ischemia (AMI) is caused by considerable intestinal injury, which is associated with intestinal ischemia followed by reperfusion. To elucidate the mechanisms of ischemia/reperfusion injuries, a C5a inhibitory peptide termed AcPepA was used to examine the role of C5a anaphylatoxin, induction of inflammatory cells, and cell proliferation of the intestinal epithelial cells in an experimental AMI model. In this rat model, the superior mesenteric artery was occluded and subsequently reperfused (Induce‐I/R). Other groups were treated with AcPepA before ischemia or reperfusion. Induce‐I/R induced injuries in the intestine and AcPepA significantly decreased the proportion of severely injured villi. Induce‐I/R induced secondary receptor for C5a‐positive polymorphonuclear leukocytes in the vessels and CD204‐positive macrophages near the injured site; this was correlated with hypoxia‐induced factor 1‐alpha‐positive cells. Induction of these inflammatory cells was attenuated by AcPepA. In addition, AcPepA increased proliferation of epithelial cells in the villi, possibly preventing further damage. Therefore, Induce‐I/R activates C5a followed by the accumulation of polymorphonuclear leukocyte and hypoxia‐induced factor 1‐alpha‐producing macrophages, leading to villus injury. AcPepA, a C5a inhibitory peptide, blocks the deleterious effects of C5a, indicating it has a therapeutic effect on the inflammatory consequences of experimental AMI.

Targeting Mitochondrial Dysfunction with L-Alpha Glycerylphosphorylcholine.

Background We hypothesized that L-alpha-glycerylphosphorylcholine (GPC), a deacylatedphosphatidylcholine derivative, can influence the mitochondrial respiratory activity and in this way, may exert tissue protective effects. Methods Rat liver mitochondria were examined with high-resolution respirometry to analyze the effects of GPC on the electron transport chain in normoxic and anoxic conditions. Besides, Sprague-Dawley rats were subjected to sham operation or standardized liver ischemia-reperfusion (IR), with or without GPC administration. The reduced glutathione (GSH) and oxidized glutathione disulfide (GSSG), the tissue myeloperoxidase, xanthine oxidoreductase and NADPH oxidases activities were measured. Tissue malondialdehyde and nitrite/nitrate formation, together with blood superoxide and hydrogen-peroxide production were assessed. Results GPC increased the efficacy of complex I-linked mitochondrial oxygen consumption, with significantly lower in vitro leak respiration. Mechanistically, liver IR injury was accompanied by deteriorated mitochondrial respiration and enhanced ROS production and, as a consequence, by significantly increased inflammatory enzyme activities. GPC administration decreased the inflammatory activation in line with the reduced oxidative and nitrosative stress markers. Conclusion GPC, by preserving the mitochondrial complex I function respiration, reduced the biochemical signs of oxidative stress after an IR episode. This suggests that GPC is a mitochondria-targeted compound that indirectly suppresses the activity of major intracellular superoxide-generating enzymes.