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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online April 8, 2003 as doi:10.1096/fj.02-0918fje. |
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* Phospholipid GmbH, Cologne, Germany and Institute of Surgical Research, University of Szeged, Szeged, Hungary
2Correspondence: Institute of Surgical Research, University of Szeged, P.O. Box 464, H-6701 Szeged, Hungary. E-mail: boros{at}expsur.szote.u-szeged.hu
SPECIFIC AIM
Recent evidences suggest that reductive stress rather than oxidative stress is a common cause of abnormal biological oxygen radical activity. We hypothesized that under such conditions, electrophilic methyl groups (EMGs) bound to positively charged nitrogen or sulfur moieties may act as protective electron acceptors and that this poising mechanism may entail the generation of methane gas.
PRINCIPAL FINDINGS
1. To test this theory, we designed pilot experiments with isolated rat liver mitochondria in which the possible formation of methane was explored
In this system, mitochondria were incubated in gas-tight vials and reductive stress was generated by hypoxia and/or adding several reducing agents (including ascorbic acid, NADPH, NADH, dithiothreitol, reduced glutathione, N-acetyl-L-cysteine, etc). The rate of methane formation was determined by gas chromatography with flame ionization detectors. The results confirmed the presence of methane: the formation was linearly related on the amount of mitochondria incubated, amount of hydrogen peroxide added (0–20 mM), and the pH of the reaction mixture. Acidic pH increased methane generation, but there was a significant methane evolution even at pH 7.0 (see inset graphs in Fig. 1
).
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2. Catalase completely abolished the increase in methane production, which indicates that mitochondrial hydrogen peroxide is required for the hypoxic activation of the methane-generating reaction
Dimethylthiourea, pyruvate, and mannitol were less effective (
80% inhibition was observed); addition of superoxide dismutase did not affect methane generation.
3. To interpret the findings and to clarify the probable mechanisms, it became necessary to study the interaction of the key reactants in isolation, i.e., in simple buffers containing no mitochondria or other biological structures
We conducted a series of in vitro studies with the components assumed to be present in the mitochondria and to play a role in generating reductive stress. Each step in the postulated reaction pathway was studied systematically; methane formation as well as the possible evolution of other gases was monitored in several concentration ranges and in different ratios of the components (electron acceptor EMGs, hydrogen peroxide, reducing agent, and Fe3+ ions).
We determined the relative effectiveness of potential methyl group donor compounds (choline chloride, betaine, phosphatidylcholine) and reducing agents (ascorbate, NADH, NADPH, dithiothreitol, N-acetyl-l-cysteine) in terms of methane generation. The most potent ones, ascorbate and choline chloride, were used in further experiments. Experiments were performed in gas-tight vials connected to a syringe. The composition of the gas phase was analyzed by gas chromatography.
4. In all experimental series, the reactions started immediately with visible gas formation, color changes, and an elevation of temperature
Methane generation increased linearly with the amount of hydrogen peroxide, choline added and the concentration of catalytically active iron. With increasing ascorbate concentrations, methane generation reached maximum (50 µmol/total gas volume) at 0.5 M, then declined; the optimal ratio of oxidant/reducing agent was 0.5:1 in this setting.
5. The completed reaction could be restarted only by the addition of hydrogen peroxide, suggesting the reaction is dependent on the generation of hydroxyl radicals
6. Methane formation was significantly enhanced at acidic pH, and the pH decreases in parallel with methane generation
7. Carbon monoxide and carbon dioxide were also present in the gas phase
Carbon dioxide evolved in measurable amount within 30 s after the start of the reaction. Carbon monoxide appeared parallel to methane formation.
8. The reaction is exothermic; 45°C, 60°C, 74°C, and 95°C peak temperature was measured after 0.25M, 0.5M, 1M, and 2M H2O2, respectively
Parallel with heat generation, the gas volume increased: the expansion moved the piston of the syringe attached to the reaction tube in proportion to the amount of the generated carbon dioxide.
9. Choline concentration affected methane generation exclusively, not carbon monoxide or carbon dioxide production
Conversely, methane formation was related exclusively to the presence of choline (or other EMGs).
10. Ascorbate was the source of carbon dioxide and carbon monoxide
The same amount of carbon monoxide and carbon dioxide was formed with or without choline, which indicates that the oxidized carbon gases are the end-products of a Fenton-type reaction and their formation is independent of methane generation.
11. When the amount of the individual components was reduced, methane formation decreased linearly; the detection limit was reached in the 0.1 mM range
CONCLUSIONS AND SIGNIFICANCE
Here we report for the first time the generation of methane by rat liver mitochondria. We also report the formation of methane from choline in the presence of hydrogen peroxide, catalytic iron, and ascorbic acid. In our in vitro experiments, each step in the postulated reaction pathway was studied systematically; methane formation as well as the possible evolution of other gases was monitored in several concentration ranges and in different ratios of the components (electron acceptor EMGs, hydrogen peroxide, reducing agent, and Fe3+ ions were used, respectively). Results of the model experiments indicate that choline and ascorbate together could possess poising efficacy and relieve reductive stress. In this system, carbon dioxide and carbon monoxide are formed from the ascorbate molecule parallel to methane generation. This is summarized in Fig. 2
.
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These results suggest that an elevated reducing power could potentially not only reduce oxygen but also electron acceptor biomolecules, thus leading to the formation of methane. The formation of methane has been thought to be an exclusive attribute of methanogenic archaea, producing methane from decomposing organic matter in anaerobic freshwater environment and in the gastrointestinal tract of mammals. The formation of methane by aerobic cells or organelles has hitherto not been reported. In animal mitochondria, the formation of methane cannot be an energy gaining catabolic step, but solely the outcome of a process that lowers the excessive reducing equivalent.
It seems reasonable to assume that generation of carbon monoxide and carbon dioxide in these reactions is the result of the oxidative breakdown of ascorbic acid, whereas the generation of methane from choline reflects an increasingly reductive (negative) redox potential in the system as a whole. The latter is probably also reflected by the invariable pH shift toward the acid side. The parallel occurrence of a violent oxidation sequence (here of ascorbic acid) and a violent exergonic shift in redox potential activating a poising response (here the generation of methane from choline) may be directly relevant to an essential biological protective mechanisms.
While the auto-oxidation of ascorbic acid has been extensively studied, this interaction, which may provide a model of reductive stress generated in biological systems (and to the protective response to it), has not been described previously. All components of the initial reaction mixture (other than free iron) are now known to be ubiquitous constituents in comparatively high concentrations of biological systems. Although the in vivo significance of the reaction requires further study, this poising role could explain the still incompletely understood essential role of choline in the diet and its preventive efficacy in a number of experimentally induced pathologies associated with redox imbalance. The mechanistic details of such a pathway suggest a new explanation of the loss of methyl groups in pathologies with abnormally elevated mitochondrial NADH/NAD+ ratio. The electron transfer will result in the formation of measurable amounts of methane perhaps through a surviving ancient evolutionary trait. Similarly, the generation of carbon monoxide is particularly interesting in view of recent reports of its possible biological signaling and protective role.
A steady state of reducing power (redox balance) may be as important for the normal functioning of aerobic cells as is a constant pH. Conversely, redox imbalance may be as common and important a feature of abnormal clinical states as is acid base imbalance. The two are in fact linked. Attention has focused in the past on "oxidative stress". We believe that the reverse imbalance, "reductive stress," is far more common and potentially life-threatening. We can consider reductive stress (the rise in the concentration of reductive equivalents) comparable to acidosis as a prime threat to homeostasis. Like acidosis, reductive stress can be envisaged as a possible or even inevitable consequence of numerous abnormal metabolic processes, including impairment of the respiratory chain by hypoxia. It could occur in a wide and important range of clinical conditions such as iron overload and diabetes. Although buffering and carbon dioxide evolution alone can undoubtedly mitigate reductive stress (since pH and redox potential are linked), this is almost certainly inadequate when reductive stress is severe or when it occurs in conjunction with an acid shift. The defense mechanism that may operate in biological systems against such reductive stress may be the capture of electrons by EMGs and the consequent irreversible evolution of methane gas. We propose that this rather than antioxidants is probably the main protective mechanism against oxygen free radical damage.
FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-0918fje; to cite this article, use FASEB J. (April 8, 2003) 10.1096/fj.02-0918fje 
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