A RAT COLONIC RING MODEL TO STUDY H2 AND CH4 PRODUCTION IN VITRO

Abstract

Breath H2 and CH4 tests are being developed to assess intraintestinal function, based on the assumption that intestinal bacteria utilize the exogenous substrates to produce these gases. To determine in vitro conditions affecting production in the presence and absence of exogenous substrate, washed isolated rat colonic rings were incubated under N2 at physiologic pH and temperature in closed flasks and the production of H2 and CH4 determined. In the absence of exogenous substrate, negligible (H2<20 ppm, CH4<1 ppm) gas was detected by 1 hour. However, high concentrations (H2>100 ppm, CH4>2 ppm) were detected after 24 hrs of incubation. With the addition of lactose, dramatically increased H2 production occurred at 1 and 24 hrs; CH4 production was only increased by lactose addition after 24 hrs. H2 production occurred at pH 7.0, while CH4 occurred between 4 to 6. The increased production of gases was associated with 10,000 fold increases in bacterial colony counts on the colonic rings and in the media, as well as 200 fold increases in acetate concentration in the media.Conclusions: These results suggest that gas production in colonic ring preparations is subject to quantitative changes in bacteria, pH and metabolite formation analogous to in vivo conditions. In addition, bacteria firmly attached to colonic tissue appear to utilize colonic mucosa to support their own growth in the absence of exogenous substrate.