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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.

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.

The claim of methane (CH4) formation in plants has caused much controversy and debate within the scientific community over the past 4 years. Here, using both stable isotope and concentration measurements, we demonstrate that CH4 formation occurs in plant cell cultures that were grown in the dark under sterile conditions. Under non-stress conditions the plant cell cultures produced trace amounts [0.3-0.6 ng g -1 dry weight (DW) h -1 ] of CH4 but these could be increased by one to two orders of magnitude (up to 12 ng g -1 DW h -1 ) when sodium azide, a compound known to disrupt electron transport flow at the cytochrome c oxidase (complex IV) in plant mitochondria, was added to the cell cultures. The addition of other electron transport chain (ETC) inhibitors did not result in significant CH4 formation indicating that a site-specific disturbance of the ETC at complex IV causes CH4 formation in plant cells. Our study is an important first step in providing more information on non-microbial CH4 formation from living plants particularly under abiotic stress conditions that might affect the electron transport flow at the cytochrome c oxidase in plant mitochondria.

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