Preliminary Studies Demonstrating Acetoclastic Methanogenesis in a Rat Colonic Ring Model

Washed rat colonic rings were incubated in closed flasks under N2 at physiologic pH and temperature levels. In the absence of an exogenous substrate, negligible H2 but some CH4 concentrations were detected in vitro after one hour of incubation, but high concentrations (H2 > 100？ppm, CH4 > 10？ppm) of both gases were found after 24 hours of culture. Production of H2 and CH4 by the washed colonic rings was stimulated by lactose addition. Maximum H2 production occurred at about pH 7.0, while maximum CH4 production occurred between pH 4.0 and 6.0. The increased production of both gases at 24 hours was associated with dramatic increases (104-fold) in anaerobic bacteria colony counts on the colonic rings and in the incubation media, as well as dramatic increases (100-fold) in acetate concentrations in the media, while lactate concentrations first rose and then fell significantly. These results suggest that gas production in colonic ring preparations is subject to quantitative changes in microbiota, pH, and metabolite formation analogous to in vivo conditions. In addition, microbiota firmly attached to colonic tissue appears to utilize colonic tissue to support its growth in the absence of an exogenous substrate. 1. Introduction Humans and animal breath contains gaseous materials not found in normal air; these materials are thought to be products of bacterial and nonbacterial metabolism [1–3]. Two of these gases, hydrogen (H2) and methane (CH4), have attracted considerable attention in recent years, [4, 5] including a study suggesting that obese patients who have methane in their breath have significantly higher body mass indexes which may play a role in obesity [6–8]. Breath H2 has been shown (a) to be reduced with fasting, (b) to be responsive to exogenous substrate ingestion, (c) to vary with the various populations, (d) to become elevated in some disease states, (e) to be subjected to lung excretion capacity, (f) to be dependent on the presence of bacteria, and (g) to show a close response to colonic bacterial content. By contrast, breath CH4 appears (a) to be unresponsive to carbohydrate substrate ingestion in adults (but not necessarily in infants), (b) to be produced at a constant rate, (c) to not be present universally in all populations, (d) to be elevated in disease states (especially cancer), and (e) to be produced by the distal colonic bacteria. Considerable attention has been devoted toward clinical applications for the measurement of breath H2 and CH4 levels. These studies have focused on H2 and CH4 generation in vivo by human or animal models or