Excessive alcohol consumption induces methane production in humans and rats

Abstract

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.

Figure 1

Exhaled CH₄ production profile of humans during the course of the study. Exhaled CH₄ production on days 1–5 and 31–33 of the investigation is depicted. During the first measurement on day 1, all volunteers were untreated. Alcohol consumption induced significant CH₄ production by the 2^nd day compared to the 1^st day, and this level decreased remarkably by the 5^th day. Mean values and standard error of mean (SEM) are given; **p < 0.01 and ***p < 0.001 vs. day 1. The arrows pointing upwards and downwards represent start and end of alcohol intake, respectively. Statistics: one-way ANOVA with Dunnett’s multiple comparison test.

Figure 2

Liver-specific enzyme levels before and after alcohol consumption in humans. Enzyme levels were determined in plasma from blood samples taken on days 1 (control, left boxes) and 5 (last day of alcohol consumption, right boxes) of the investigation. During the first measurement, all volunteers were untreated. (A) Aspartate aminotransferase (AST), (B) Alanine aminotransferase (ALT), (C) Alkaline phosphatase (AP) and (D) Gamma glutamyl transferase (GGT) levels. Alcohol consumption did not alter enzyme levels. Mean values and standard error of mean (SEM) are given; p < 0.05 vs. day 1 (control). Statistics: Paired-test.

Figure 3

Whole-body CH₄ release of rats. Whole body CH₄ production was measured on days 1, 3, 5, and 8 of the investigation. Ethanol feeding induced significant CH₄ production by the 3^rd day in the 1.15 g/kg/day alcohol-treated group and by the 5^th day in the 2.7 g/kg/day alcohol-treated group, compared to control group. However, in the GPC-treated groups no increase was observed as compared to controls. Empty circles with continuous line relates to control group, black triangles with continuous line to the 1.15 g/kg/day alcohol-treated group, empty triangles with dashed line to the 1.15 g/kg/day alcohol + GPC-treated group, black squares with continuous line to 2.7 g/kg/day alcohol-treated group, and empty squares with dashed line to the 2.7 g/kg/day alcohol + GPC-treated group. During the measurement on day 1, all the rats were untreated. Mean values and standard error of mean (SEM) are given; *p < 0.05, **p < 0.01 and ***p < 0.001 vs. Control group, ^###p < 0.001 vs. corresponding GPC-treated groups. Statistics: Two-way ANOVA, Bonferroni post-hoc test.

Figure 4

Oxygen consumption of rat liver mitochondria. O₂ consumption rate of mitochondria was assessed in liver homogenate after addition of 0.5 μM rotenone, 10 mM succinate and 2.5 mM ADP on day 9 of investigation. The 2.7 g/kg/day ethanol-fed group had decreased mitochondrial function compared to control, whereas the GPC treatment prevented the decline of mitochondrial oxygen consumption, demonstrated by higher levels compared to both non-GPC treated ethanol-fed groups. Mean values and standard error of mean (SEM) are given; **p < 0.01 vs. Control, ^#p < 0.05 vs. corresponding GPC-treated groups. Statistics: One-way ANOVA with Bonferroni post-hoc test.

Figure 5

Oxygen consumption of rat hippocampus mitochondria. O₂ consumption rate of mitochondria was assessed in hippocampus homogenate after addition of 0.5 μM rotenone, 10 mM succinate and 2.5 mM ADP on day 9 of investigation. Mitochondrial respiratory capacity deteriorated upon alcohol intake regardless of GPC treatment. Mean values and standard error of mean (SEM) are given; ***p < 0.001 vs. Control. Statistics: One-way ANOVA with Bonferroni post-hoc test.

Figure 6

Liver-specific enzyme levels before and after alcohol consumption in rats. Enzyme levels were determined in plasma from blood samples taken on days 1 (untreated control, left boxes) and 8 (last day of alcohol treatment, right boxes) of the investigation. The 1.15 g/kg/day alcohol (panels A–C) and 1.15 g/kg/day alcohol + GPC-fed group (panels D–F) were monitored with same method. Both Aspartate aminotransferase (AST, panels A and D) and Alanine aminotransferase (ALT, panels B and E) levels increased significantly as compared to baseline. Alkaline phosphatase (AP, panels C and F) levels did not change. Mean values and standard error of mean (SEM) are given; *p < 0.05 vs. day 1 (Control). Statistics: Paired t-test.